Knight was born in Marylebone, London. She is the daughter of Esmond Knight and his first wife, Frances Clare, and the stepdaughter of Actress Nora Swinburne. Being from a theatrical family, she was introduced to theatre at an early age. She was inspired by a visit to the bombed-out Old Vic Theatre in 1949 with her father to see performances of The Snow Queen and As You Like It. After studying there for two years under Glen Byam Shaw and George Devine, she was offered a position as Assistant Stage Manager at the Midland Theatre Company in Coventry. From Coventry, she moved to Ipswich Repertory Company where Joe Orton was a fellow ASM.

Two years later she joined a touring group, the West of England Theatre Company, for an eight-month stint. She was spotted by a Producer, which led to her being cast as a schoolgirl in Blue Murder at St Trinian's in 1957. That same year, she starred with her father, playing father and daughter, in the BBC production of Nicholas Nickleby. An earlier film role, albeit uncredited, was as a lady-in-waiting in Laurence Olivier's film Richard III (1955), which also starred her father. She performed in two early Carry On films. In Carry On Nurse, she played Nurse Nightingale and in Carry On Teacher she played Felicity Wheeler, a prim school inspector whose amorous hopes toward Kenneth Connor's wimpy science master are continually thwarted.

Knight married Director and Producer Michael Elliott in July 1959 and they were married until his death in 1984. They had two daughters – Marianne Elliott, a theatre Director, and Susannah Elliott-Knight. Marianne is married to actor Nick Sidi and they have a daughter, Eve, born in August 2004.

In 1963, she played Mrs Fitzpatrick in the film of Tom Jones and was in a second St. Trinian's film, playing a Teacher in The Wildcats of St. Trinian's in 1980.

She has made countless appearances on television, in shows such as Mapp & Lucia (1985), Only Fools and Horses (1989), Agatha Christie's Poirot (1992), Jeeves and Wooster (1993), The Upper Hand (1995), Wycliffe (1996), Dalziel and Pascoe (1999), Heartbeat (2000), Casualty (2002), Midsomer Murders (2003 and 2011), Doctors (2005 and 2009), Agatha Christie's Marple (2006), Holby City (2008 and 2015), Sherlock (2012) and Friday Night Dinner (2012 and 2016). From 1999 to 2001 she co-starred in the sitcom Gimme Gimme Gimme, playing Beryl Merit, the retired prostitute landlady of the two main characters, Linda (Kathy Burke) and Tom (James Dreyfus).

Her other films include Prick Up Your Ears (1987) and About a Boy (2002). Throughout her career, Rosalind Knight has continued to work in the theatre, including with the Royal Shakespeare Company, the Royal Court Theatre and the Old Vic. She also worked at the Manchester Royal Exchange and the Sheffield Crucible.