Gubler was born on March 9, 1980, in Las Vegas, Nevada, the son of Marilyn (née Kelch), a rancher and political consultant, and John Gubler, an attorney. Gubler is a high school graduate of the Las Vegas Academy of International Studies, Visual and Performing Arts, where he majored in acting, because the school did not offer his first choice, filmmaking. He is a graduate of New York University's Tisch School of the Arts, where he majored in film directing.
While at NYU film school, Gubler was found by a model scout and then worked as a model with DNA Model Management for Tommy Hilfiger, Marc Jacobs, and American Eagle, among others. He was ranked 46th on models.com list of top 50 male models.
After he began modeling, Gubler had an internship with Wes Anderson, who encouraged him to audition for a part in his movie The Life Aquatic with Steve Zissou. Gubler did, and landed the role of Nico (a.k.a. Intern #1). This then led in 2005 to his leading role as Dr. Spencer Reid in Criminal Minds.
Gubler had a small role in the film RV (2006) as the disreputable Joe Joe. He voiced the character of Simon (the smart Chipmunk) in Alvin and the Chipmunks (2007), Alvin and the Chipmunks: The Squeakquel (2009), Alvin and the Chipmunks: Chipwrecked (2011), and Alvin and the Chipmunks: The Road Chip (2015) alongside Jason Lee (Dave), Justin Long (Alvin), and Jesse McCartney (Theodore). He appeared as Bart, the main character, in How to Be a Serial Killer (2008), and as Paul in (500) Days of Summer (2009). He provided the voice of Winsor in Scooby-Doo! Legend of the Phantosaur (2011). He has also provided on/off voice-work for Bagdasarian Productions (regarding the character Simon), whenever Ross Bagdasarian Jr. was unable to voice the character himself. This is most notable in the episode "Who's The Animal?".
In 2014, Gubler acted in the zombie comedy Life After Beth and the film Suburban Gothic, for which he received the Screamfest Award for Best Actor. He also played Joe Harper in the 2015 movie Band of Robbers.
Gubler has done voice work for the DC Universe Animated Original Movies line. He played Jimmy Olsen in All-Star Superman and voices The Riddler in the animated film: Batman: Assault on Arkham (2014). Gubler has also since appeared in the film Band of Robbers (2015).
While making The Life Aquatic with Steve Zissou, Gubler made a documentary titled Matthew Gray Gubler's Life Aquatic Intern Journal, a behind-the-scenes view of the making of the film, which was later included in The Criterion Collection DVD release. He has directed and starred in a series of self-deprecating mockumentaries named Matthew Gray Gubler: the Unauthorized Documentary in which he parodies Hollywood behavior, which are filmed while on the set of Criminal Minds. A follow-up Matthew Gray Gubler: The Authorized Documentary was also made, although only one episode is currently available.
Gubler directed, edited, and co-produced the music video for "Don't Shoot Me Santa" by the rock band The Killers.
Gubler directed the Criminal Minds season 5 episode "Mosley Lane" (5x16), which originally aired March 3, 2010, along with the episode "Lauren" (6x18), which aired a year later on March 16, 2011. The episode "Lauren" included the exit of Criminal Minds series regular Paget Brewster, who maintains a friendship with Gubler outside of work on the show. Brewster specifically asked Gubler to direct her last episode, although it was announced later that she would return for season 7. Gubler has since directed the Criminal Minds episodes "Heathridge Manor" (7x19), which aired April 4, 2012, "The Lesson" (8x10), which aired December 5, 2012, "Alchemy" (8x20), "Gatekeeper"(9x07), "Blood Relations" (9x20), "Mr. Scratch" (10x21), "A Beautiful Disaster" (11x18), which included the departure of series regular Shemar Moore, and "Elliott's Pond" (12x06).
Since 2005, Gubler has been posting paintings on his website. His media include watercolor, gouache, oil, and pastel. In September 2005, the Gallery of Fine Art in Ostrava, Czech Republic, held a showing of 12 watercolors by Gubler; all the works were sold. On September 6, 2008, his art was featured in a group show entitled "Paper Cuts" at The Little Bird art gallery in Atwater Village, California. Gubler contributed a painting of "Sandy The Mammoth" to the Western Heritage Museum & Lea County Cowboy Hall of Fame. In July 2010, his paintings were featured in Juxtapoz magazine.
In October 2011, an original watercolor by Gubler entitled "Mushface" was sold at auction site eBay for $10,100. Gubler donated the proceeds to the Smith Center for the Performing Arts in Las Vegas.
Gubler's paintings were highlighted in a 2013 interview with BuzzFeed.
Gubler divides his time among Los Angeles, Las Vegas, and New York City.
In 2009, Gubler dislocated his knee while dancing. The injury required three surgical procedures and the use of a cane for nearly a year.
On October 27, 2014, Gubler became a certified minister and officiated at the wedding of his best friends Criminal Minds co-star Paget Brewster and Steve Damstra, on November 29, 2014.