Born in Dearborn, Michigan, Burtka grew up in Canton, Michigan, and graduated from Plymouth Salem High School in 1993. He trained in acting at Interlochen Center for the Arts, obtained his Bachelor of Fine Arts at the University of Michigan and had further training at the William Esper Studios.

His Broadway debut was as Tulsa in the 2003 production of Gypsy starring Bernadette Peters. He played The Boy in the American premiere of Albee's The Play About the Baby, for which he won the 2001 Clarence Derwent Award for most promising male performer.

Burtka made his television debut in 2002 with a guest role on The West Wing; this was followed by guest appearances on Crossing Jordan and How I Met Your Mother. The actor recently reprised his role as "Scooter" (whose actual first name is Bill), the former high school boyfriend of Lily (Alyson Hannigan) in the penultimate Second Season episode of How I Met Your Mother.

In 2004, Burtka originated the role of Matt in the musical The Opposite of Sex, and reprised the role in the work's east coast premiere in the summer of 2006.

Six months after Burtka's How I Met Your Mother appearance, www.canada.com published a story purporting that the actor received the part because of a romantic relationship with one of the series' stars, Neil Patrick Harris. Speculation around this story eventually led Harris to publicly acknowledge his homosexuality in a cover story in "People" magazine. Burtka made no public response to the story, though later, Harris stated that he and Burtka were moving in together.

Burtka and Harris attended the Emmy awards in September 2007 as an openly acknowledged couple for the first time, an appearance which Harris discussed on Ellen.

On February 4, 2008, Burtka and Harris appeared for the first time on stage together singing a duet from Rent at a benefit for The LGBT Community Center in New York. The two young actors have been together since 2004. Neil refers to David as "My better half" and "an amazing chef."