Kelley was delivered by his uncle at his parents' home in Toccoa, Georgia, the son of Clora (née Casey) and Ernest David Kelley, who was a Baptist minister. DeForest was named after the pioneering electronics engineer Lee de Forest. He later named his Star Trek character's father "David" after his own father. Kelley had an older brother, Ernest Casey Kelley. Kelley was immersed in his father's mission in Conyers and told his father that failure would mean "wreck and ruin". Before the end of his first year at Conyers, Kelley was regularly putting to use his musical talents and often sang solo in morning church services. Eventually, this led to an appearance on the radio station WSB AM in Atlanta. As a result of Kelley's radio work, he won an engagement with Lew Forbes and his orchestra at the Paramount Theater.
In 1934, the family left Conyers for Decatur, Georgia. He attended the Decatur Boys High School, where he played on the Decatur Bantams baseball team. Kelley also played football and other sports. Before his graduation in 1938, Kelley got a job as a drugstore car hop. He spent his weekends working in the local theaters.
During World War II, Kelley served as an enlisted man in the United States Army Air Forces from March 10, 1943 to January 28, 1946, assigned to the First Motion Picture Unit. After an extended stay in Long Beach, California, Kelley decided to pursue an acting career and relocate to southern California permanently, living for a time with his uncle Casey. He worked as an usher in a local theater in order to earn enough money for the move. Kelley's mother encouraged her son in his new career goal, but his father disliked the idea. While in California, Kelley was spotted by a Paramount Pictures scout while doing a United States Navy training film.
Kelley's acting career began with the feature film Fear in the Night in 1947. The low-budget movie was a hit, bringing him to the attention of a national audience and giving Kelley reason to believe he would soon become a star. His next role, in Variety Girl, established him as a leading actor and resulted in the founding of his first fan club. Kelley did not become a leading man, however, and he and his wife, Carolyn, decided to move to New York City. He found work on stage and on live television, but after three years in New York, the Kelleys returned to Hollywood.
In California, he received a role in an installment of You Are There, anchored by Walter Cronkite. He played ranch owner Bob Kitteridge in the 1949 episode "Legion of Old Timers" of the television series The Lone Ranger. This led to an appearance in Gunfight at the O.K. Corral as Morgan Earp (brother to Burt Lancaster's Wyatt Earp). This role led to three movie offers, including Warlock with Henry Fonda and Anthony Quinn. In 1957, he had a small role as a Southern officer in Raintree County, a Civil War film directed by Edward Dmytryk, alongside Elizabeth Taylor, Montgomery Clift and Lee Marvin. He also starred in the lead role as a U.S. Navy submarine captain in World War II in The Silent Service. He appeared in both season 1, episode 5, "The Spearfish Delivers", as Commander Dempsey and in the first episode of season 2, "The Archerfish Spits Straight", as Lieutenant Commander Enright. Leonard Nimoy also appeared in two different episodes of the series at around the same time.
Kelley appeared three times on Gunfight at the O.K. Corral: first in 1955, where he portrayed Ike Clanton in the television series You Are There; again, two years later in the 1957 film of that name, playing Morgan Earp; and perhaps in an ironic salute, once again on October 25, 1968, in a third-season Star Trek episode titled "Spectre of the Gun", this time portraying Tom McLaury.
Kelley also appeared in episodes of The Donna Reed Show, Perry Mason, Wanted: Dead or Alive, Boots and Saddles, Dick Powell's Zane Grey Theater, Death Valley Days, Riverboat, The Fugitive, Lawman, Bat Masterson, Have Gun - Will Travel and Laredo. He appeared in the 1962 episode of Route 66, "1800 Days to Justice" and "The Clover Throne" as Willis. He had a small role in the movie The View from Pompey's Head.
For nine years, Kelley primarily played villains. He built up an impressive list of credits, alternating between television and motion pictures. However, he was afraid of typecasting, so he broke away from villains by starring in Where Love Has Gone and a television pilot called 333 Montgomery. The pilot was written by an ex-policeman named Gene Roddenberry, and a few years later Kelley would appear in another Roddenberry pilot, Police Story (1967), that was again not developed into a series.
Kelley also appeared in at least one radio drama, Suspense, where series producer William M. Robson introduced him as "a bright new luminary in the Hollywood firmament".
In 1956, nine years before being cast as Dr. McCoy, Kelley played a small supporting role as a medic in The Man in the Gray Flannel Suit in which he utters the diagnosis "This man's dead, Captain" and "That man is dead" to Gregory Peck. Kelley appeared as Lieutenant Commander James Dempsey in two episodes of the syndicated military drama The Silent Service, based on actual stories of the submarine section of the United States Navy. In 1962, he appeared in the Bonanza episode titled "The Decision", as a doctor sentenced to hang for the murder of a journalist. The judge in this episode was portrayed by John Hoyt, who later portrayed Dr. Phillip John Boyce, one of Leonard McCoy's predecessors, on the Star Trek pilot "The Cage". In 1963, he appeared in The Virginian episode "Man of Violence" as a "drinking" cavalry doctor with Leonard Nimoy as his patient. (Nimoy's character did not survive.) Perhaps not coincidentally, the episode was written by John D. F. Black, who went on to become a writer-producer on Star Trek. Just before Star Trek began filming, Kelley appeared as a doctor again, in the Laredo episode "The Sound of Terror".
After refusing Roddenberry's 1964 offer to play Spock, Kelley played Dr. Leonard "Bones" McCoy from 1966 to 1969 in Star Trek. He reprised the character in a voice-over role in Star Trek: The Animated Series (1973–74), and the first six Star Trek motion pictures (1979 to 1991). In one of the Star Trek comic books it was stated that Dr. McCoy's father had been a Baptist preacher, an idea that apparently originated from Kelley's background. In 1987, he also had a cameo in "Encounter at Farpoint", the first episode of Star Trek: The Next Generation, as by-that-time Admiral Leonard McCoy, Starfleet Surgeon General Emeritus. Several aspects of Kelley's background became part of McCoy's characterization, including his pronunciation of "nuclear" as "nucular".
Kelley became a good friend of Star Trek cast mates William Shatner and Leonard Nimoy, from their first meeting in 1964. During Trek's first season, Kelley's name was listed in the end credits along with the rest of the cast. Only Shatner and Nimoy were listed in the opening credits. As Kelley's role grew in importance during the first season he received a pay raise to about $2,500 per episode, and received third billing starting in the second season after Nimoy. Despite the show's recognition of Kelley as one of its stars he was frustrated by the greater attention that Shatner received as its lead actor, and Nimoy received because of "Spockamania" among fans.
Shy by his own admission, Kelley was the only cast member of the original Star Trek series program never to have written or published an autobiography; however, the authorized biography From Sawdust to Stardust (2005) was written posthumously by Terry Lee Rioux of Lamar University in Beaumont, Texas. Kelley regarded "The Empath" as his favourite Star Trek television episode.
After Star Trek, Kelley found himself a victim of the very typecasting he had so feared. In 1972, he was cast in the horror film Night of the Lepus. Kelley thereafter did a few television appearances and a couple of movies but essentially went into de facto retirement other than playing McCoy. By 1978 he was earning up to $50,000 ($184,000 today) annually from appearances at Star Trek conventions. Like other Star Trek actors, Kelley received little of the enormous profits that the franchise generated for Paramount, until Nimoy, as executive producer, helped arrange for Kelley to be paid $1 million for Star Trek VI: The Undiscovered Country (1991) which would eventually be his final live-action film appearance. He also appeared in the very first Star Trek: The Next Generation episode, "Encounter at Farpoint", in which he portrayed a 137-year-old Dr. McCoy.
In a TLC interview done in the late 1990s, Kelley jokingly said one of his biggest fears was that the words etched on his gravestone would be "He's dead, Jim." Reflecting this, Kelley's obituary in Newsweek magazine began: "We're not even going to try to resist: He's dead, Jim." On the other hand, he stated that he was very proud to hear from so many Star Trek fans who had been inspired to become doctors as a result of his portrayal of Dr. McCoy. For his final film, Kelley provided the voice of Viking 1 in the 2nd/3rd installment in the children's series The Brave Little Toaster Goes to Mars.
Later in life, Kelley developed an interest in poetry, eventually publishing the first of two books in a series, The Big Bird's Dream and The Dream Goes On – a series he would never finish.
Kelley died of stomach cancer on June 11, 1999, at the Motion Picture & Television Country House and Hospital in Woodland Hills, Los Angeles. His body was cremated and the ashes were scattered in the Pacific Ocean.