Highmore was born on 14 February 1992 in Camden Town, London, into a show business family. His mother, Sue Latimer, is a talent agent whose clients include actors Daniel Radcliffe and Imelda Staunton, and his father, Edward Highmore, is an actor. He has a younger brother named Bertie. His home is in Highgate, North London. Highmore was educated at a primary school in Hampstead Garden Suburb in Hampstead and at Highgate School, an independent school, followed by Emmanuel College at the University of Cambridge, where he earned a Double First in Spanish and Arabic.
In 1999, Highmore made his big screen debut in the comedy Women Talking Dirty (1999), portraying Helena Bonham Carter's son. His next prominent role was in the family drama Two Brothers (2004). Highmore's breakout role came when he was cast in Finding Neverland (2004), alongside Johnny Depp and Kate Winslet. He was then cast in the title role of Charlie Bucket in Tim Burton's Charlie and the Chocolate Factory (2005), and was reportedly recommended by co-star Johnny Depp. Roles in A Good Year (2006) and Arthur and the Invisibles (2006) soon followed, before he portrayed the title role in August Rush (2007), opposite Keri Russell and Robin Williams. In 2013, he began starring as the iconic Norman Bates in the drama thriller series Bates Motel (2013), alongside Vera Farmiga as his mother. Highmore received much acclaim for his performance throughout the series' run, and was nominated for multiple Saturn Awards and Critics' Choice Television Awards.
Highmore is fluent in French and Spanish, and semi-fluent in Arabic. He got married in 2021.
He and actor Matthew Mandzij, who played the father of the boy injured at the airport in the series premiere, also previously acted together on Bates Motel. The latter played Deputy Jeffcoat, who arrests Norman in Season 2 after the death of Jimmy Brennen.
He received his first ever Golden Globe nomination for his role on this show during the first season but ultimately lost out to actor Sterling K. Brown.