Harris was born in Albuquerque, New Mexico, and grew up in Ruidoso, New Mexico. His parents, Sheila and Ron, ran a restaurant. He attended La Cueva High School in Albuquerque and was active in school plays and musicals there. Harris was an honors student, and graduated with honors in 1991.[citation needed]

Harris began his career as a child actor, and was discovered by playwright Mark Medoff at a Drama Camp in Las Cruces, New Mexico. Medoff cast him in his 1988 film Clara's Heart, a drama with Whoopi Goldberg based on the novel (of the same name) by Joseph Olshan, that won him a Golden Globe nomination. In 1988, he also starred in Purple People Eater, a children's fantasy. The following year, he won the lead in Doogie Howser, M.D., for which he was again nominated for a Golden Globe. After Doogie Howser's four-season run ended in 1993, Harris played a number of guest roles on television series such as Murder She Wrote, before taking his first film role as an adult in 1995 in the little-seen shocker Animal Room. Since then, his film work has included supporting roles in The Next Best Thing, Undercover Brother and Starship Troopers. In the Harold and Kumar stoner comedy films, (Harold & Kumar Go to White Castle and Harold & Kumar Escape from Guantanamo Bay) he plays a drug-crazed, lecherous version of himself.

From 1999 to 2000, Harris starred with Tony Shalhoub in the sitcom Stark Raving Mad, which lasted 22 episodes. He has taken lead roles in a number of made-for-television features, including Snowbound: The Jim and Jennifer Stolpa Story in 1994, My Ántonia in 1995, The Christmas Wish in 1998, Joan of Arc in 1999, The Wedding Dress in 2001, and The Christmas Blessing in 2005, as well as series guest roles.

Harris has worked on Broadway in both musical and dramatic roles. He played Tobias Ragg in 2001 concert performances of Sweeney Todd. In 2002, he performed on Broadway beside Anne Heche in Proof. In 2003, he took the role of the Emcee in Cabaret, alongside Deborah Gibson and Tom Bosley. As a result of his critically acclaimed performance in Cabaret, Harris was named the top-drawing headliner in the role of the Emcee by GuestStarCasting.com, topping fellow celeb stars John Stamos and Alan Cumming.

In 2004, he performed a dual role of the Balladeer and Lee Harvey Oswald on Broadway in the controversial musical revival of Stephen Sondheim's Assassins. He also sang the role of Charles (first played by Anthony Perkins) on the Nonesuch recording of Sondheim's Evening Primrose. He has also portrayed Mark Cohen in the touring company of the musical RENT, a character who he mockingly reprised on the January 10, 2009, episode of Saturday Night Live, which he hosted. Since fall of 2005 Harris has played Barney Stinson, a serial womanizer reminiscent of his Harold and Kumar role, in the CBS ensemble sitcom How I Met Your Mother. The Stinson role earned him 2007, 2008, 2009 and 2010 Primetime Emmy Award for Outstanding Supporting Actor in a Comedy Series nominations.

In 2007, Harris worked with Mike Nelson on an audio commentary for RiffTrax. The two riffed on the film Willy Wonka And The Chocolate Factory. Harris is a big fan of the cult TV series Nelson worked on, Mystery Science Theater 3000, and was interviewed for a 1992 Comedy Central special hosted by Penn Jillette, who did voiceovers for Comedy Central's programming at that time, about the series and its fans, This Is MST3K. In 2008 Harris took the title role in Joss Whedon's musical web series, Dr. Horrible's Sing-Along Blog, alongside Nathan Fillion and Felicia Day. The first episode of the series debuted July 15, 2008. In 2008 Harris also guest-starred on Sesame Street as the Sesame Street Fairy Shoe Person. On April 26, 2009, Harris hosted the 7th Annual TV Land Awards.

After a highly successful preview at the San Diego Comic-Con, a musical episode of Batman: The Brave and the Bold, featuring Harris as the villainous Music Meister, premiered on October 23, 2009, on Cartoon Network. As a character who could make anyone do his bidding by singing, he spent most of the episode singing several original songs.

Harris stars in the upcoming movie Beastly, alongside Alex Pettyfer and Vanessa Hudgens. He plays a blind tutor named Will. The film was originally set for release on July 30, 2010, but was pushed back to March 18, 2011.

He also plays the lead in the upcoming indie comedy The Best and the Brightest as a carefree father who uproots his family from Delaware to move to New York City's Upper East Side.

Harris appeared as a guest judge on Season 9 of American Idol in August 2009 auditions taped in Dallas, Texas.