Urie then studied at Collin County Community College before being accepted at the Juilliard School in New York City. There he was a member of the Drama Division's Group 32 (1999–2003), which also included Jessica Chastain. Urie graduated from Juilliard in 2003.
Urie, while still a student at Juilliard, performed in the world premiere of Love and Happiness (2001) at the Consolati Performing Arts Center, starring as a sixteen-year-old trying to get rid of his mother's boyfriend. In addition to this, he appeared in student productions of Sylvia (1998) and Locked Away (1999) at Quad C Theatre.
He received the 2002 John Houseman Prize for Excellence in Classical Theatre from the Juilliard School. His classical credits include Shakespeare, Jacobean drama, and commedia dell'arte.
Starting in 2006, Urie began appearing in ABC "dramedy" Ugly Betty, appearing as Marc St. James, the assistant of Wilhelmina Slater (Vanessa L. Williams). The show began with the concept that Wilhelmina would have a different assistant in each episode, thus Urie was originally billed as a guest star in the credits, However, Williams loved their chemistry, and Urie was signed on as a full-time regular midway through the first season. He and the cast were nominated for Screen Actors Guild awards for Outstanding Performance by an Ensemble in a Comedy Series in 2007 and 2008. The role earned Urie a Ewwy Award nomination for Best Supporting Actor in a Comedy Series in 2009. He remained with Ugly Betty until the show's cancellation in 2010.
During the 2007–08 Writers Guild of America strike, Urie hosted TLC's reality-based series Miss America Reality Check. The program followed the contestants participating in the 2008 Miss America Pageant.
On October 29, 2008, he appeared as a guest presenter on the British National Television Awards, awarding for the Best Performance in a Serial Drama.
In 2009, Urie referred to himself as "a member of the LGBT community" on his website. In a 2010 interview with The Advocate, he said that he was in a relationship with a man and identifies as "queer". He said it never felt wrong when he was with women previously.
Urie is one of the leads in CBS's half-hour pilot Partners. The multi-camera comedy, from Will & Grace creators Max Mutchnick and David Kohan, centers on lifelong friends and Business partners – one straight and one gay. The pilot also features Sophia Bush as another lead. The series premiered on September 24, 2012, but was cancelled after only six episodes had aired.
His performance in 2013's one-man show Buyer & Cellar won him a Clarence Derwent Award.
In April 2015, Urie became the host of Cocktails & Classics on Logo TV, in which he and panels of Celebrity friends watch and comment on classic movies while imbibing cocktails named or made for the films. Films profiled on the series have included All About Eve, Steel Magnolias, Valley of the Dolls, Breakfast at Tiffany's, and Mommie Dearest.
In November 2018, Urie will be starring as Arnold Beckoff in Harvey Fierstein's Torch Song revival on Broadway.
