She was born on August 10, 1972 in Highland Park, Texas, the daughter of Daphne Demar (née Caravageli) and Lawrence Paul "Larry" Harmon, a hospital information network executive in Dallas, Texas. Her father has German and Irish ancestry, while her mother is of Greek descent. In an episode of Who Do You Think You Are?, Harmon found that she had a German ancestor who served in George Washington's army during the Revolutionary War, and that ancestor later acquired land in Mercer County, Kentucky. The land is still owned by a distant branch of her family.
Harmon worked as a child model, and in 1987 won a Seventeen modeling contest. She attended Highland Park High School until 1990, where she was a member of the Highland Belles. She won a Spectrum Model Search contest shortly afterward, subsequently pursued a successful modeling career, and became well known in the early 1990s. She worked as a runway model for Calvin Klein, Giorgio Armani, and Donna Karan and appeared on the covers of ELLE, Cosmopolitan, and Esquire. She is signed with IMG Models in New York City.
Harmon began acting in 1995 after being discovered on a plane by David Hasselhoff. She then starred in several television series, including Baywatch Nights, Baywatch, and the short-lived C-16. She also appeared in the 1998 film, Lawn Dogs, which received only a limited theatrical release. In the late 1990s, Harmon became better known by joining the long-running NBC series Law & Order, playing ADA Abbie Carmichael from 1998 to 2001. During this time, she also voiced Barbara Gordon in the animated film Batman Beyond: Return of the Joker, succeeding Stockard Channing in that role.
Harmon left Law & Order to concentrate on her film career, saying that she preferred working in film to television. After appearing in the 2001 direct-to-video film Good Advice, she had a supporting role in Agent Cody Banks (2003), playing the CIA handler of a teenage agent (Frankie Muniz). In 2006, Harmon co-starred with Cuba Gooding Jr. and James Woods in the direct-to-DVD political suspense drama End Game.
In 2006, Harmon starred in an ABC pilot Secrets of a Small Town, which did not go to series. She also starred in another failed television show on NBC, Inconceivable, which was canceled after two episodes. The following year, she starred in another ABC pilot, Women's Murder Club. The police drama was picked up, but only ten of the thirteen episodes aired.
Harmon was one of five actresses who posed discreetly nude for the May 2008 issue of Allure magazine, alongside Gabrielle Union, Zoe McLellan, Jill Scott, and Ana Ortiz.
In 2010 Harmon hosted an infomercial for "UpLiv", a stress management program and also appeared in an infomercial for Olay "Pro-X" wrinkle cream.
From 2010 to 2016, she co-starred with Sasha Alexander in the TNT crime-drama series Rizzoli & Isles, playing Boston, Massachusetts police detective Jane Rizzoli. The show premiered July 12, 2010. She directed the series' 100th episode.
Harmon appears in the music video for Craig Morgan's "This Ole Boy", released in January 2012.
In July 2012, during promotions for a new season of Rizzoli and Isles, Harmon revealed on Conan O'Brien's late night series that she was a fan of and very interested in playing a movie version of the She-Hulk from Marvel Comics should such a movie become available.
Harmon won Outstanding Female Actor in a Leading Role in a Drama Series for Rizzoli & Isles at the 2012 Gracie Allen Awards.
On March 13, 2000, Harmon became engaged to former NFL player Jason Sehorn. She was appearing as a guest on The Tonight Show with Jay Leno when Leno called Sehorn out as a surprise guest. Sehorn immediately walked up to her, knelt down on one knee and proposed. They were married on June 9, 2001. They have three daughters: born October 2003, June 2005, and December 2008. In November 2014, Harmon and Sehorn announced their separation after 13 years of marriage. As of 2016, the couple are now divorced.
Harmon is a Republican. She and Sehorn supported President George W. Bush and delivered a speech at the 2004 Republican National Convention. She supported John McCain for president in 2008. Harmon is a devout Christian and has stressed the importance of prayer in her daily life.