Purefoy was screen tested for the role of James Bond in 1995 for GoldenEye, but ultimately lost the role to Pierce Brosnan. Throughout 2004 and 2005 Purefoy's name was rumoured as a possible candidate to replace Brosnan as Bond in future films. He played James McCarthy, a young man accused of murdering his father, in "The Boscombe Valley Mystery," in Granada'sThe Case-Book of Sherlock Holmes. One of his notable roles was as Nicholas Jenkins in the eight-part miniseries A Dance to the Music of Time for Channel 4 in 1997. He played Edward, the Black Prince in the film A Knight's Tale, Rawdon Crawley in Vanity Fair with Reese Witherspoon and Tom Bertram in the 1999 production of Mansfield Park. He has played major roles in several television costume dramas, including Sharpe's Sword, The Tenant of Wildfell Hall, The Prince and the Pauper, The Mayor of Casterbridge, Blackbeard: Terror at Sea, , The Tide of Life, Camelot and Rome. He was originally the actor for V in the 2006 Film V for Vendetta but had creative differences with the production team and left the film six weeks into filming. Parts of the film contain scenes of Purefoy. Speculation suggested that his departure was due to an opportunity to play James Bond in the 2006 film Casino Royale. He played Mark Antony in the HBO/BBC original television series, Rome. At the time there were rumours that at least one nude body in the show had been digitally enhanced. When his Wikipedia entry, which at that time referred to the rumours, was brought up in an interview with Alastair McKay, published in the January 2007 issue of Out magazine, Purefoy said, "I won't say whose it was, but there was a penis in the series that may have been slightly enhanced. But it wasn't mine. Mine's all mine." Producer William J. MacDonald announced that James Purefoy would play Simon Templar in a new TV series of The Saint. The new series was scheduled to start shooting in Berlin and Australia in April 2008. However, production ultimately did not occur and in August Purefoy was reported as negotiating with NBC to star in another series, The Philanthropist. He starred as Teddy Rist in the summer television series, The Philanthropist, which aired on NBC beginning on June 2009. His character is a billionaire playboy who decides to use his wealth and power to help others in need. In 2013, The Following debuted, starring Purefoy as the lead antagonist of the series. He portrays Joe Carroll, a former professor who becomes a serial killer and leads a cult of followers, all whom help create Carroll's "story". It was announced in June 2014 that Purefoy would join the cast of the film High-Rise with Tom Hiddleston and Jeremy Irons. Later in 2014, Purefoy co-starred in the Formula 1-themed music video for David Guetta's song "Dangerous". In February 2019, Hybrid released a short film to accompany their single "Hold Your Breath" from the albumLight of the Fearless. The film starred Purefoy as the brooding 'Mr Black'.
Purefoy has a son, Joseph, with the actress Holly Aird. He also has a daughter, Rose and another two sons, Ned and Kit, with documentary director and producer Jessica Adams. He married Adams in Somerset in July 2014. Purefoy is a supporter of Yeovil Town FC.