Serafinowicz was born 10 July 1972 in Liverpool. He is of Belarusian and Polish descent. He attended Our Lady of the Assumption Primary School and St Francis Xavier Secondary School.

Serafinowicz made his broadcasting debut in 1993 on Radio 1 show The Knowledge, a spoof documentary about the music industry. From there he went on to perform in Radio 4 shows Week Ending, Harry Hill's Fruit Corner, Grievous Bodily Radio, The Two Dannys and A Whole New Ball Game.

On 1 May 1997, he appeared in The Election Night Armistice as Moz Bingham, the fictional press secretary to the then Shadow Chancellor of the Exchequer Gordon Brown, who verbally abuses and bullies the BBC's Nick Robinson in a spoof phone call over the politician's news coverage. In 1998 he appeared on TV in Comedy Nation and You are Here. He also had a role in the Simon Nye-penned sitcom How Do You Want Me?.

Serafinowicz made guest appearances on television, including the comedy shows Smack the Pony and Hippies (both 1999), Black Books (2000), I'm Alan Partridge (2002) and Little Britain (2003), comedy drama Murder Most Horrid (1999) and ITV drama series Agatha Christie's Marple (2005).

In 2001, he took the lead in BBC Two sitcom World of Pub, playing the same character as he had done in the show's run on Radio 4. He appeared in The Junkies, an Internet sitcom. In 2002, the critically acclaimed Look Around You, a series of 10-minute 1970s school science video spoofs, debuted on BBC Two in which he played a scientist. He co-wrote the show alongside Robert Popper, whom he met on the set of Spaced. The show returned in 2005 as a spoof of the 1980s show Tomorrow's World, with Serafinowicz playing Peter Packard, one of the presenters.

In 2003, he appeared in another Nye-written sitcom, Hardware, in which he played the affable Kenny. He reprised this role in the second series in 2004. Also in 2004 he appeared in British rom-zom-com (romantic-zombie-comedy) Shaun of the Dead, playing Pete, the tidy flatmate of principal characters Shaun and Ed. He appeared in boxing film The Calcium Kid.

A second O! News video later appeared, parodying the 2006 Apple Corps v. Apple Computer lawsuit. This attracted the interest of the BBC, and a pilot was commissioned for The Peter Serafinowicz Show. A full series was broadcast on BBC Two in 2007 (with a Christmas special in 2008). For his performance, Serafinowicz was presented with the Best Entertainer prize at the 2008 Rose d'Or ceremony. The series was also nominated for Best Comedy Programme at the 2009 BAFTA Television Awards.

In October 2007, Serafinowicz dropped his attempt to use the Human Rights Act against the national newspapers. He had sought to prevent the publication of details revealing that his grandfather, Szymon Serafinowicz, was the first man in the UK to have been tried under the War Crimes Act. His grandfather was charged with direct involvement in three murders and personal involvement in the destruction of the Jewish populations of Mir and Minsk, but found unfit for trial on grounds of dementia in 1997 and died later that year, aged 86.

In 2008, Serafinowicz was working with Robert Popper on a new television series, as well as a website, based on a spoof religion "Tarvuism" for US network Adult Swim. In 2009, he appeared in Universal Films' Couples Retreat as Sctanley, the manager of the Eden Resort, directed by Peter Billingsley.

Due to his uncanny, near-perfect impersonation of ex-Beatle Paul McCartney, Matt Berry sang a duet with him on Berry's 2009 album Witchazel. The track in question was "Rain Came Down", where he was billed as "Paul Mcartney" (sic).

In 2010, he co-starred with Will Arnett in the FOX sitcom Running Wilde, devised by Arrested Development creator Mitchell Hurwitz. It premiered on Fox on 21 September 2010, but was cancelled by the network after 13 episodes.

In June 2013, Serafinowicz provided the English language audiobook recording for the Roald Dahl novel Danny, the Champion of the World. In July 2013, he made his film directing debut with the dark comedy film I See What You Did There, which he wrote, along with Danny Wallace.

In 2014 it was announced that Serafinowicz would play the role Crowley in the BBC Radio 4 adaption of Terry Pratchett and Neil Gaiman's novel Good Omens.

He stars in the title role of Amazon's The Tick, the pilot for which premiered on 19 August 2016.