London Mayor Sadiq Khan said the Central and Victoria lines will start on 19 August, with the Piccadilly, Jubilee and Northern lines following in the autumn.
The service will run through the night on Fridays and Saturdays.
It was due to start last September but was delayed due to disputes with unions.
Last year union members went on strike over the introduction of the all-night service, as well as their pay and conditions.
The service will run:
Maintenance workers belonging to the RMT union are still in dispute with Transport for London. The union agreed a deal for its drivers in March, but said the deal for maintenance staff was "inferior".
About 200 part-time drivers are currently taking part in a 14-week training programme for the new service.
RMT general secretary Mick Cash said there were "major" unresolved issues to do with conditions and pensions, and that it still had major concerns over safety.
"Against a background of massive cuts overshadowing TfL budgets all parties have to be clear that Night Tube, a development that RMT supports, cannot be delivered on the cheap," he said.
The mayor said: "The Night Tube is absolutely vital to my plans to support and grow London's night-time economy - creating more jobs and opportunities for all Londoners.
"The constant delays under the previous mayor let Londoners down badly. I have made getting the Night Tube up and running a priority."
London's transport commissioner, Mike Brown, said: "More than half a million people use the Tube after 10pm on Fridays and Saturdays, and the introduction of the Night Tube, which will support London's businesses and jobs, is a historic step in our modernisation of the Underground and our work to support London's economic growth."
Of all of the previous mayor Boris Johnson's schemes, the Night Tube was the most problematic to get off the ground.
I once called it a zombie policy - half dead wandering around, causing nothing but trouble.
It was meant to start in September 2015 but the transport unions were not happy about pay and changes to their work life balance.
The eventual solution - as well as bonuses - was to hire in part-time night drivers.
Now we have a start date (again) but crucially it is only for the Central and Victoria lines. That's because there are still unresolved issues with RMT staff who work mainly on the other planned night lines - the Jubilee, the Northern and the Piccadilly.
By going ahead with a start anyway there is a risk that antagonises the unions. It could be the first big test for the new mayor's relationship with London's transport unions.
Until it's running on five lines, there are probably more twists to come.
A date has been set for the launch of the Night Tube service - almost a year after it was first scheduled to begin.