One of Braff's earliest roles was in High, a proposed 1989 CBS television series with a cast that also included Gwyneth Paltrow and Craig Ferguson; the television pilot never made it on air. Braff appeared in the 1990s series The Baby-sitters Club, in an episode titled "Dawn Saves the Trees". He appeared in Woody Allen's 1993 film Manhattan Murder Mystery. In 1998, Braff had a part in a George C. Wolfe production of Macbeth for New York City's Public Theater.

Braff has wanted to be a filmmaker since his early childhood, and has described it as his "life dream." Braff was diagnosed with obsessive-compulsive disorder at age ten. During his childhood, Braff was a friend of Future Fugees member Lauryn Hill. As a child, Braff also attended Stagedoor Manor, a performing arts "training center" for youth actors ages 10 to 18. It was there that he won an acting award and had his first kiss. Stagedoor is also where Braff met actor Josh Charles, who is still a friend. Braff also knows Stagedoor alums Natalie Portman, Mandy Moore, and Joshua Radin. Braff attended the Northwestern University School of Communication, where he became a brother of the Phi Kappa Psi fraternity, and graduated in the class of 1997.

Braff played John "J.D." Dorian on the medical comedy TV series Scrubs which debuted in 2001. The role was Braff's first major role in a television show. Braff was nominated for three Golden Globes and an Emmy for his work on the show. Braff directed several episodes of Scrubs, including its one-hundredth, "My Way Home". For the show's ninth season, Braff was a cast member for six episodes and also served as one of the executive producers.

Braff's is expected to direct Open Hearts, a remake of the 2002 Danish film Elsker dig for evigt (Love You Forever). The film is about a woman who has an affair with her paralyzed husband's Doctor, whose wife caused the accident that put her husband in a wheelchair. It was first revealed that Braff was directing Open Hearts in 2006, however, the movie was cancelled. Braff said "It fell apart at the last second due to scheduling and budget, as so many movies do". At the same time that Braff's film The Last Kiss was being released.

Braff began dating fellow actor, Mandy Moore, in 2004. They ended their relationship in 2006 after two and half years of dating.

In Episode 4.3 of the MTV show Punk'd, which aired on March 20, 2005, Braff was filmed yelling at a teenaged actor. He unwittingly appeared on the show at the behest of his Scrubs co-star, Donald Faison, who pranked Braff by having an actor spray-paint Braff's new Porsche with fake paint. Braff pursued and caught the teen actor and tried to punch him in the stomach; this was edited out of the episode.

Braff starred in the romantic drama The Last Kiss, which opened on September 15, 2006. Braff tweaked several parts of Paul Haggis' script for the film, as he wanted the script to be as "real as possible" and "really courageous" regarding its subject matter. As with Garden State, Braff was involved with the film's Soundtrack, serving as executive Producer. The film's Director, Tony Goldwyn, compared Braff to a younger version of Tim Allen, describing Braff as "incredibly accessible to an audience... a real guy, an everyman."

In 2007, Braff starred in the film The Ex (2007), which he has described as a "silly comedy".

In November 2008, Braff earned his pilot's license flying a Cirrus SR20.

In 2009, Braff opened up the restaurant Mermaid Oyster Bar in New York City with chef and high school friend Laurence Edelman.

Braff starred in the Canadian indie film The High Cost of Living with Quebec Actress Isabelle Blais in 2010. Directed by Deborah Chow, the film was shot in Montreal and principal photography wrapped on March 9, 2010. Braff stated he enjoyed filming in the country in which The Last Kiss was also shot. The film premiered at the Toronto International Film Festival and was also shown at the Tribeca Film Festival.

In early 2011, Braff announced that he had written a play to be performed at the Second Stage Theatre in mid-2011. The play, All New People, is set on Long Beach Island and centers on Charlie, a 35-year-old from Braff's home state New Jersey. The play was directed by Peter DuBois, who directed Braff in Trust the previous year. When announcing the play on Facebook, Braff wrote that 'one of my dreams comes true'. In 2012, Braff moved the play on tour to the UK, playing in Manchester at the Manchester Opera House between February 8–11, Glasgow at the King's Theatre between February 14–18, and finally in London for 10 weeks at the Duke of York's Theatre from February 22.

Proprietors of the Rio Theater in Monte Rio, California credited Braff with making the donation that put their Kickstarter campaign over the target to buy a digital projector over its $60,000 goal in May 2013.

On April 10, 2014, Braff opened on Broadway in the musical Bullets Over Broadway The Musical, an adaptation of Woody Allen's 1994 film, directed and choreographed by Susan Stroman.