She was born in 1940 Osterley, as the daughter of Basil John Nightingale (1906-) and his wife, Celia nėe Winter. After attending Lady Eleanor Holles School, Hampton, southwest London, and the Polytechnic of Central London (now the University of Westminster) School of Journalism, Nightingale began her career as a journalist in Brighton, East Sussex. In the 1960s and 1970s, she wrote columns for the Daily Express, the Daily Sketch, Petticoat and Cosmopolitan magazine.
Her first broadcast on the BBC was on 14 September 1963 as a panellist on Juke Box Jury, and she contributed to Woman's Hour in 1964 and hosted programmes on the BBC Light Programme in 1966.
She started at Radio 1 on 5 October 1969 with a Sunday evening show. Later she then hosted the singles review show What's New in the early 1970s before graduating to a late-night progressive rock show, which was simulcast on the Radio 2 FM frequency.
In the mid-late 1970s, she presented a Sunday afternoon request show, and in the early 1980s she presented a Friday night show and the non-music-based Radio 1 Mailbag and Talkabout.
In 1978, Nightingale became the main presenter of The Old Grey Whistle Test on BBC2 as a replacement for long-time host Bob Harris. During her tenure, the show moved away from its traditional bias under Harris towards country music, blues rock and progressive rock and embraced popular modern styles such as punk rock and new wave. She left the series in 1982.
She had begun The Sunday Request Show in September 1975, originally on Sunday afternoons until the end of 1979. It began its second and most famous run in December 1982, for most of its run in a slot immediately after the Top 40. The show was one of the first on British radio to regularly play music from CDs, taking advantage of its FM carriage before Radio 1 had its own higher-quality frequencies. A gimmick was to allow the intro of the first song in the show to play uninterrupted before saying "Hi" in the very last second before the vocals started.
In 1994, Nightingale moved to a weekend overnight dance music show initially called The Chill Out Zone. She can still be heard in the early hours of Wednesday mornings on BBC Radio 1 and 1Xtra. On her current show, she spins breaks, often featuring major breaks DJs such as Plump DJs, Freestylers, Noisia and Meat Katie. Nightingale regularly DJs live at clubs and festivals around the UK and Europe.
As a DJ, Nightingale has travelled all over the world and made music-documentaries during visits to Russia, Romania, Iraq, Chile, The Philippines and Cuba. While in Havana in 1996, she was injured during a mugging, resulting in multiple injuries requiring an air-lift to a London hospital, since which she has worn the distinctive shades, now part of her image.
In 2002, Nightingale was appointed as a Member of the Order of the British Empire for her services to radio broadcasting and awarded The Caner Of The Year Award by Muzik Magazine. The award recognised her in depth coverage of the radio scene. In 2004, she was the first female DJ from Radio 1 to be inducted into the Radio Academy Hall Of Fame.
Nightingale has published two autobiographical books: Chase The Fade (1981) ISBN 0-7137-1167-1 and Wicked Speed (1999) ISBN 0-283-06197-9. She has compiled three albums: Annie On One (1996, Heavenly Recordings), her own installment of the Breaks DJ mix series Y4K (2007, Distinctive Records), and 'Masterpiece' on the Ministry of Sound compilation series of that name (July 2015)
On 30 September 2007, to celebrate the 40th anniversary of BBC Radio 1, Nightingale co-hosted a special return of the Request Show with Annie Mac featuring contributions from musicians such as Paul McCartney and Chemical Ed, excerpts from the original show and Nightingale's recollections of regular contributors such as "Night Owl of Croydon". The show featured many classic tracks which had been requested over the years and closed with one of Nightingale's favourites, Cristina's version of "Is That All There Is?".
A version of The Smiths song "Panic" interpreted by Mancunian cult comedian Frank Sidebottom dedicates its choruses to "Anne the deejay" and asks "Anne Nightingale what's your blinking game". In 2014, she appeared in The Life of Rock with Brian Pern as herself.
On 20 May 2011, she played the lead role in the UK TV Movie Annie Nightingale: Bird on the Wireless, documenting her life and passion for music.
In 2013, Nightingale was featured in the BBC Radio 4 programme Getting on Air: the Female Pioneers, presented by Jane Garvey.
In 2015, it was revealed that Nightingale had been approached by the BBC to sign a letter warning Prime Minister David Cameron that his plans to reform the corporation would damage it. Nightingale, one of the letter's 29 signatories, revealed later on that she had not read the letter prior to signing it.
In April 2012, Annie presented a show on BBC Radio 2 called "Annie Nightingale's Eternal Jukebox". She has continued presenting this on an occasional basis usually on bank holidays. The Eternal Jukebox showcases "enjoyably unexpected musical pairings." Listeners are invited to suggest a song and Annie pairs it up with another song often of a different genre and suggests a link between the two songs. On 25 June 2012, she also presented a documentary for BBC Radio 2 called "Is It Worth It?" about the Falklands Conflict. It was described on the Radio 2 website as "30 years on from the Falklands conflict, Annie Nightingale considers the impact of the war through the song Shipbuilding."
Towards the end of November 2013, it was announced that Annie Nightingale would return to BBC Radio 2 on 1 January 2014 for another one off show entitled, "Annie Nightingale: Whatever Next?". This was a 2-hour show broadcast between 8pm and 10pm on New Year's Day 2014. Annie curated "seven decades of unmissable music from the 1950s through to today." The show featured a variety of genres from the seven decades from the 1950s onwards.