Kirkby did not originally intend to become a professional singer. In the late 1960s, while she was studying classics at Oxford, she joined the Schola Cantorum of Oxford, a student choir which, at the time, was being conducted by Andrew Parrott. After graduation, Kirkby went to work as a school teacher, but became increasingly involved in singing with the growing number of music ensembles that were being founded during the Early music revival of the early 1970s. She married Parrott, and sang with his Taverner Choir which he founded in 1973. Her vocal career developed throughout the 1970s, and she became noted as a soloist in performances and recordings with prominent early music performers, including Anthony Rooley and the Consort of Musicke and Christopher Hogwood’s Academy of Ancient Music.

In 1994, Kirkby was awarded an Honorary Degree (Doctor of Music) from the University of Bath. In 1999 she was voted ‘Artist of the Year’ by Classic FM Radio listeners and in November 2000 she received the Order of the British Empire. BBC Music Magazine in April 2007 published a survey of critics to nominate “The 20 greatest sopranos”, placing Kirkby at number 10.

She was appointed a Dame Commander of the Order of the British Empire in the 2007 Queen’s Birthday Honours List. In 2010 she became President of Dartington Community Choir. On 21 January 2011 it was announced that Kirkby had been awarded the Queen’s Medal for Music, an award funded by the Privy Purse and given to an individual who has had a major influence on the musical life of the nation.

From 1971–83, Kirkby was married to conductor Andrew Parrott. She was later in a relationship with lutenist Anthony Rooley, with whom she has a son. On 30 April 2015 she married conductor Howard Williams.

In 2018, Kirkby was awarded the REMA Early Music Award in recognition of her career as an artist and mentor to young Early Music performers. In 2019, she was awarded the Lifetime Achievement Award at the Gramophone Classical Music Awards ceremony.