Services between 1am and 5.30 am will begin on the Central and Victoria lines from 27 November.
Thousands signed a petition demanding its resumption in the wake of Sarah Everard’s murder, which highlighted the issue of women’s safety.
Mayor of London Sadiq Khan said the Night Tube would “make a huge difference to people travelling around our city at night”.
“I know how important this is to London’s thriving night-time economy, to London’s recovery and to the confidence and safety of everyone travelling home at night, particularly women and girls,” he added.
“I am determined to make our city as safe as possible for all Londoners.”
Khan said the Central and Victoria lines were selected “because they’re the busiest” and “people have more confidence using the busiest lines”.
Other lines will reopen once enough staff are available, he added.
The Night Tube, which first began in August 2016 and ran on selected lines on Fridays and Saturdays, was halted when lockdown began last year.
Services were suspended because drivers were needed for daytime services.