Lord Bailey of Paddington raised the idea of a vote in the capital in the Lords.
The Tory peer, who challenged Mr Khan for the London mayoralty in 2021, said: "For the last few years, at the insistence of a Labour mayor, Londoners in council tax bands D and B have had an extra £60 added to their bill to pay for Transport for London.
"Yet the mayor is about to enter negotiations with the unions for a four-day week and an inflation-busting pay rise."
He added: "What is the referendum policy for Council Tax for London?
"With the charges that the mayor keeps heaping on people and these raises in mind, will the Government ask him to give taxpayers in London an opportunity to have their voices heard?"
Transport minister Baroness Taylor of Stevenage responded: "My Lords, the citizens of London had a chance to express their view in the recent election for the Mayor of London, and they did so resoundingly."
A spokesman for Mr Khan later added: "Lord Bailey is woefully out of touch with Londoners.
"A London-wide referendum would potentially cost Londoners tens of millions of pounds, money that should be spent on public services.
"The Mayor has had to raise council tax in recent years to make up for years of chronic underfunding by the previous Tory Government, and to provide more vital investment for the police."
The Mayor's council tax precept for 2024/25 is £471.40 for a Band D property.
The Standard revealed earlier this month that Tube drivers were being offered a new deal including a four-day week and paid meal breaks in return for calling off strikes.
Aslef, the union that represents most Tube drivers, stressed the changes mean that they will be at work for 2.5 hours a week less than at present.
Aslef had threatened to close the London Underground on November 7 and 12 unless TfL made an improved offer to address what the union called "exhausting" shift patterns.
LU agreed by January to "set out a proposal for delivering an average four-day working week with a paid meal relief included in working hours which means fewer hours at work, whilst improving the reliability and efficiency of our service and maintaining the current 35-hour contractual working week".
The Standard was told that, in practice, it would mean drivers would take their 30-minute meal break during paid "company time" - enabling them to go home after eight hours at work rather than 8.5 hours.
TfL sources said the four-day week would only be introduced if it could be sure that this would not affect the reliability of Tube services.
The transport body is understood to have not made a commitment for a four-day working week on London's transport network, and a reduction in contractual hours will not form part of any proposal.