His first film was as the main character in The Stranger's Hand starring Trevor Howard in 1953. Possibly his earliest television work was the part he played in the Sherlock Holmes episode of "The Unlucky Gambler" broadcast on 18 July 1955. However, his earliest recognised work was a part in the film It's Great to Be Young (1956) where he appeared alongside John Mills. He appeared alongside Keith Michell and Belinda Lee in the opulent swashbuckler, Dangerous Exile (1957) as Louis XVII, the ten-year-old son of Louis XVI and Marie Antoinette. Also during this period he featured in two episodes of the television series The Adventures of Robin Hood (1957) alongside Richard Greene, including as Will Dale in the episode "The Challenge of the Black Knight", as well as in the same company's Sword of Freedom series (1957) playing Alberto in the episode "Chart of Gold". He also acted in one Carry On film, Carry On Teacher (1959) in the small role of student Robin Stevens. Around the same time, he was cast in the role of Pierre van der Mal who appears in an early scene of The Nun's Story (also 1959), in which he bids farewell to his sister Gabrielle (Audrey Hepburn) as she leaves home to enter the convent. Also around this time he had a leading role in an episode of the Sapphire Films/ITC series The Four Just Men ("The Man With The Golden Touch", 1959), as Neapolitan street urchin Pietro who foils a robbery.

For the remainder of the 1960s, he was a jobbing actor appearing in such TV series as Dr. Syn: the Scarecrow, Emergency Ward 10, Redcap, Danger Man, No Hiding Place, Dixon of Dock Green and Strange Report among others, until he was offered the role of Dr. Lawrence Bingham in the LWT sitcom Doctor at Large (1971), a role which continued in the later Doctor in Charge (1972–73). Meanwhile, he also had a main role in the Thames Television comedy Alcock and Gander (1972) with Beryl Reid.

In 1973, he starred as Robin Tripp, a trainee chef, in the flatshare sitcom Man About the House written by Johnnie Mortimer and Brian Cooke. When the series ended in 1976, he continued playing Robin Tripp in the spin-off sitcom Robin's Nest, in which Robin sets up a bistro with funding from his girlfriend Vicky's (Tessa Wyatt) father James Nicholls (Tony Britton).

In 1979 he starred in the title role of LWT's young person's drama series Dick Turpin, which ran until 1982. He then played the widower Simon Harrup in the sitcom Me and My Girl broadcast from 1984 to 1988, co-starring Tim Brooke-Taylor and Joan Sanderson and also produced by LWT. He also appeared in a one-off comedy-drama The Giftie, shown on Channel Four in 1988, in which he and a friend discovered a photocopier at work that could duplicate living copies of themselves, and unwisely set about doing so, predictably leading to mistaken identities and chaos. In the 1990s, his profile decreased although he was never short of work. His final acting role was in a 1996 one-off satire entitled Holed with Tony Robinson about a suburban golf club.

Robin's Nest was a big success and the first UK sitcom to feature an unmarried couple cohabiting. To tie-in with the series, he wrote a recipe book called Man About the Kitchen, which was published in 1980. He also wrote the Robin's Nest theme tune, which was arranged by Brian Bennett. During this period, O'Sullivan also appeared in adverts for British Gas.

O'Sullivan largely retired from public life in 1996. His last appearance on television was as a guest on a 1999 edition of This Is Your Life held in honour of his Doctor... co-star George Layton. O'Sullivan had himself been the subject of the show in 1974.

He has lived in Brinsworth House, a retirement home for entertainers, since suffering a stroke in 2003.

In 2006, O'Sullivan recorded a commentary for the DVD release of Carry On Teacher.