Macfarlane was born Thomas Luke MacFarlane, Jr. in London, Ontario. His father, Thomas (deceased), was the Director of Student Health Services at the University of Western Ontario, and his mother, Penny, is a mental health nurse at a London hospital. Macfarlane attended London Central Secondary School with twin sister Ruth and older sister Rebecca.
Macfarlane studied drama at Juilliard in New York City, where he starred in the productions of Romeo & Juliet, Richard III, The School of Night, Blue Window, The Grapes of Wrath, and As You Like It before graduating from the drama division in 2003.
Macfarlane was one of the four leads in Juvenilia at the Playwrights Horizons Theater from November 14–December 21, 2003. He played the lead role in the American premiere of the play Where Do We Live, staged at the Vineyard Theatre in May 2004. The production was cited by the 2005 GLAAD Media Awards for Outstanding New York Theatre: Broadway and Off-Broadway. He also appeared with Jill Clayburgh and Hamish Linklater in the off-Broadway production of The Busy World is Hushed, again at Playwrights Horizons, in Summer 2006. He reprised his role of Thomas for the L.A. Premiere at the Skirball Cultural Center from February 7-11 of 2007.
On television, Macfarlane is perhaps best known for his role as Pvt. Frank "Dim" Dumphy on the 2005 FX network television series Over There. He played opposite Cynthia Nixon in Robert Altman's Tanner on Tanner on the Sundance Channel.
At present, Macfarlane has a permanent role on ABC's Brothers & Sisters as Scotty Wandell, husband to one of the "brothers" of the show, Kevin Walker (played by Matthew Rhys). The character of Scotty has been the center of some debate, as his more buoyant personality (contrasting to Kevin's masculinity) has led fans and critics like TV Guide's Michael Ausiello, to be critical of what Ausiello referred to as "Kevin's swishy friend."
In the episode "Date Night" it was established that Scotty's "flamboyant" personality was an act, due to being nervous and wanting to feel less insecure. Scotty and Kevin dated for several episodes before they broke up due to Scotty feeling Kevin did not treat him with enough respect. Macfarlane received praise for his portrayal of a type of gay man who is often written and performed in very stereotypical terms. Actor Rhys said that the show received a big reaction when Kevin and Scotty broke up, and that the relationship had "struck a chord".
Luke was the last cast member to be added to the FOX pilot, Supreme Courtships, playing the role of conservative Allen Moore, described as a trust fund baby trying to prove he's more than just a legacy in the court. The pilot was not picked up by Fox Broadcasting Company for the 2007–2008 season. In his latest travail for TV, Macfarlane was cast as the leading role in the upcoming two-part mini-series called Iron Road which filmed in China for five weeks and British Columbia for two weeks. This mini was shot from late April to mid June 2007, and was set to be released in April 2008, though it has not yet aired.
Macfarlane was part of the one-night celebrity performed staging of Howard Ashman's unproduced musical "Dreamstuff". The musical was re-imagined by Howard's partners Marsha Malamet and Dennis Green and performed one night only at Los Angeles' Hayworth Theatre as part of the Bruno Kirby celebrity reading series, directed by Ugly Betty's Michael Urie. Luke starred in the show alongside Eden Espinosa, Vicki Lewis, Fred Willard and David Blue (actor).