Spencer was born in Melbourne on 12 February 1979. He was born to Rodney Spencer, a radiologist, and Robyn Spencer. He is one of four siblings. Spencer has two older brothers: Tarney Spencer, an oculoplastic surgeon, and Luke Spencer, an orthopaedic surgeon. His younger sister, Polly Spencer, is an anaesthetist. Growing up, he attended Canterbury Primary School, Malvern Central School, and the private Scotch College. While there, he auditioned for the long-running soap opera Neighbours. He completed his VCE and had gained a place at Monash University but deferred to pursue acting.

Spencer met his House M.D. co-star, American actress Jennifer Morrison, in March 2004 at Vancouver International Airport when they were on their way to film the pilot episode of House. They started dating in July 2004 and became engaged while vacationing in Paris at the Eiffel Tower during Christmas 2006, but in August 2007, Spencer and Morrison announced they had called off their engagement. Their characters were also involved on the show, marrying and divorcing in season six.

Spencer starred as Billy Kennedy in the Australian soap opera Neighbours from 1994 to 2000. He later reprised his role in 2005, in an episode celebrating the show’s 20th anniversary. He has since co-starred in films such as Winning London, Uptown Girls and Swimming Upstream. From 2004 to 2012, he starred as Dr. Robert Chase on the Fox medical drama House. He played Chase for the show’s entire run, becoming the second longest-serving member of the title character’s team. In 2012, Spencer began starring as Matthew Casey in the NBC drama Chicago Fire.

For Neighbours, Spencer was nominated for “Most Popular Actor” at the 1998 and 1999 Logie Awards. For House, he was nominated for “Choice TV Breakout Performance – Male” at the 2005 Teen Choice Awards and “Outstanding Performance by an Ensemble in a Drama Series” at the 2008 Screen Actors Guild Awards. He received a “Golden Boomerang” at the 2006 Australians in Film Breakthrough Awards for his work on House. In 2007, he was included in People Magazine’s 100 Most Beautiful People issue.

Spencer began his musical career with the Australian Boys Choir, performing with them from 1986 to 1992. He plays the violin in Band from TV, a group which includes his former House co-star Hugh Laurie. They play at various events, giving the money they earn to the different charities. The band played on Idol Gives Back in April 2008.