Labour Mayor Sadiq Khan boarded the first train as it left Brixton underground station - but was almost left on the platform after stopping for selfies.
The Night Tube start date on the Central and Victoria lines is Friday August 19.
Around 100 British Transport Police (BTP) officers are patrolling the network, which operates through the night on Fridays and Saturdays.
But Superintendent Chris Horton insisted there was "no reason" why passengers would not be as safe as during the day.
London Mayor Sadiq Khan (centre) travelling with passengers on a northbound Victoria line Tube train during the launch of the Night Tube service on Saturday.
He commented: "It's not me making a cheap point, but the previous mayor did announce the Night Tube start date on one occasion and that wasn't met; on a second occasion, that wasn't met; on a third occasion, that wasn't met; and then gave up".
"You can feel the buzz", Khan told the news service.
GETTYMayor of London Sadiq Khan rode the Tube with fellow late night travellers
Advocates of the Night Tube emphasise its benefits on the rest of the cultural sector, with theatres and galleries now able to stay open late if visitors can get home.
"I'm really pleased that 100 days or so after becoming the mayor we've got that right", Khan said.
The first underground railway service ran between Paddington and Farringdon Street in central London on January 10 1863 and now the "tube", known the world over for its red roundel symbol, stretches well into London's suburbs and beyond.
Asked if he was concerned about feeling exhausted, Daniel George, 28, from Cheshunt, Hertfordshire, said: "No, you change your sleeping pattern to work around that sort of thing".
He added: "It's great for people not having to worry about the last train".
Excited Londoners and tourists jostled to be among the first to take the Night Tube, London's new overnight subway service that plans to host 50,000 riders each weekend.
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