Amstell was born in Gants Hill, Redbridge, east London in 1979, to David and Tina (née Leventhal) Amstell. He was brought up in a Jewish family, and he was the eldest of four children. He was educated at Beal High School, a state comprehensive school in Ilford. His parents' divorce in the early 1990s has been a constant theme throughout his stand-up career, and he says that their decision when he was born to circumcise him had caused him a sense of great emotional as well as physical loss.

Amstell's first television appearance was in 1993 when he appeared as a contestant on the Channel 4 game show GamesMaster. In 1994 he appeared on Good Morning with Anne and Nick performing an impression of Dame Edna Everage. Also that year, he appeared in an episode of Family Catchphrase, describing himself as a budding Magician and showing one of his tricks to the show's presenter Andrew O'Connor, himself a Magician. Amstell and his family won a television and a video recorder.

In 1995, Amstell created Radio XL at his secondary school Beal High School which is still a big part of Beal tradition today

Amstell started performing on the comedy circuit when he was in his early teens and later became the youngest finalist to appear in the BBC New Comedy Awards. His first professional television appearance was in 1998 as a presenter on the UK children's channel Nickelodeon. He claims he was sacked for being "sarcastic and mean to children" although the posted biography on his website also, in jest, claims:

From 2000 to 2006, Amstell presented Popworld on Channel 4 with Miquita Oliver. He voiced the characters "Timothy the Popworld melon" and "Richard the Popworld horse" and developed a highly ironic, surreal and left-field style which gained the show a cult following but angered many of its guests. One Example was a mock interview with singer Lemar called "Lemar From Afar" in which Amstell shouted questions into a megaphone from one end of the world's largest car park while Lemar stood at the other end. Another was a "Si-chiatrist" interview with Luke Pritchard and Hugh Harris of The Kooks in which Amstell played the role of Psychiatrist and Pritchard and Harris his patients.

Amstell first appeared on the comedy show Never Mind the Buzzcocks as a guest during Mark Lamarr's tenure as host, in 2003 and 2006. Following Lamarr's departure, he was one of the series' guest hosts before being appointed as permanent host beginning in October 2006. He said at the time that he hoped to beat "the universal, exceptionless rule that when a new host takes over an old show it is a horrible, embarrassing disaster".

Amstell also performs as a stand-up Comedian. He has performed at the Edinburgh Festival Fringe in 2005, 2006, 2007 and 2009. In 2008, Amstell completed his first UK tour. He appeared at the Just for Laughs Comedy Festival in Montreal in July 2009. His entire run at the Edinburgh Fringe Festival 2009 at the Bongo Club sold out. He took part in his second national tour in the autumn of 2009.

Amstell co-wrote an episode of Channel 4 teenage drama Skins in 2007, titled "Maxxie and Anwar".

Amstell performed Do Nothing in 2009 at the Royal Court Theatre, raising money for their young Writers programme.

In May 2010, Amstell recorded Do Nothing in Dublin's Vicar Street venue for a November DVD release. In December 2011 and April 2012, it was shown on BBC3 and the BBC iPlayer.

In 2011, Amstell appeared in the independent film Black Pond.

On New Year's Eve 2012, a one-off stand-up special performance of Simon Amstell's stand-up show, 'Numb', which he toured to sell-out audiences in 2012, was recorded for the BBC at TV Centre.

In 2014 Simon Amstell announced a new tour of UK and North America, titled 'TO BE FREE'.

In 2015, he provided voice work for the video game Dr. Langeskov, The Tiger and The Terribly Cursed Emerald: A Whirlwind Heist, for which he received critical praise.

In 2016, Amstell hosted the Evening Standard British Film Awards.

In 2017 Amstell announced his third stand-up UK tour, titled 'What Is This?'. It began in London on the 22nd September 2017.