The Mayor said he would not risk Londoners' safety "for the sake of a few pounds" at a meeting of the London Assembly on Thursday.
His remarks cast doubt on a future for driverless trains on the London Underground.
Four years ago, former London Mayor Boris Johnson unveiled the first new-look driverless trains which he hoped would enter service on the Piccadilly line in 2022.
Mr Khan said: "Anybody who understands the basic knowledge of the underground system will know there are some deep level, deep underground services, where the idea of having driverless trains is madness.
"In the interests of saving some pounds to risk the safety of Londoners, of staff and visitors, is the height of recklessness.
"Where it's not safe to do so I will not do so. Where it's safe to do so of course we'll consider whatever efficiency savings that we can do."
Mr Johnson unveiled a range of driverless trains in 2014 for the Piccadilly line.
The RMT union said it was "deeply concerned" about the plans at the time, though Mr Johnson denied the innovations would lead to job losses.
Later that year, an internal TfL document revealed driverless trains were unlikely to be in operation on the Underground until 2030.
Mr Khan's latest statements appear to indicate that plans for unmanned Tube trains would not be given the green light anytime soon.
A spokesman for the RMT told the Standard: "It would be madness to introduce driverless trains on any part of the Underground.
"Boris Johnson may have said all kinds of things were going to happen but no politician would risk the carnage that would come with putting unmanned trains on that network.
"We are pleased to see Mr Khan make his position on the matter clear."