St Anne's Limehouse was formed from part of the parish of St. Dunstan's Stepney, prior to the 18th century a large (but then thinly populated) East London parish that extended all the way down to the Thames River. As the population of London increased, growing parishes were subdivided. In 1709 a new parish in Limehouse was formed from part of the parish of St. Dunstan. The church may be named for Queen Anne as she raised money for it by taxing coal passing along the River Thames.

The building was designed by Nicholas Hawksmoor, as one of twelve churches built to serve the needs of the rapidly expanding population of London in the 18th century. The scheme never met its original target, but those built were also known as the Queen Anne Churches. The building was completed in 1727 and consecrated in 1730.

Queen Anne decreed that as the new church was close to the river it would be a convenient place of registry for sea captains to register vital events taking place at sea. Therefore, she gave St. Anne's Church the right to display the second most senior ensign of the Royal Navy, the White Ensign. The prominent tower with its golden ball on the flagpole became a Trinity House "sea mark" on navigational charts, and the Queen's Regulations still permit St. Anne's Limehouse to display the White Ensign 24 hours a day, 365 days a year.