The British Transport Police (BTP) officers will patrol trains and all 51 stations on the Central and Victoria lines on Friday and Saturday night, Transport for London (TfL) announced today.
Officers from the Metropolitan Police Service, which is responsible for policing beyond tube stations, will be at key hubs and bus stations.
BTP rapid-response vehicles and more than 12,000 CCTV cameras across the network will support the police presence at stations.
"Passengers on the Night Tube must be able to travel with the same confidence they are used to during the day," mayor of London Sadiq Khan said.
"That's why we're investing £3.4m to ensure that dedicated officers are on hand to offer the support and visible reassurance Londoners expect."
Crime rates on London's transport network have fallen by more than 20 per cent over the last five years.
Evidence from 24-hour metros around the world suggests crime levels at night are no greater than during the day, TfL added.
City of London police superintendent Paul Clements said: "
We already have a robust, visible policing strategy across the City throughout Friday and Saturday nights, and we are confident we will be prepared and ready to attend all incidents quickly.
"However, we are not complacent and we are aware that extra Tube services are likely to mean greater footfall in the area, allowing people to visit licensed premises for longer, or to vary their working hours within the City, for instance."
The Night Tube is forecast to deliver a £3bn boost directly to the capital's economy and could create up to 115,000 extra jobs, research for EY and London First found last week.
This could rise to £43bn and 1.37m jobs by 2030 if London becomes "a truly 24-hour city".
Later this year, Night Tube services will extend to include the entire Jubilee line and much of the Northern line.
“We also plan to expand the night time service to parts of the Metropolitan, Circle, District, and Hammersmith & City lines once our modernisation programmes are complete,” TfL has said.