Khan also demanded more autonomy from central government, including on public spending, in order to "protect" the British capital's economy.
While the campaign to leave the European Union won Thursday's referendum nationwide with 52 percent, Londoners voted 60 percent in favour of Britain staying in the EU along with Scotland, Northern Ireland and Gibraltar.
London should have "a full seat at the negotiating table", Khan said at a business conference, according to a copy of his speech posted on the mayor's website.
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"Remaining in the single market needs to be priority one, two and three of our negotiation with the EU.
"As much as I might like the idea of a London city state, I'm not seriously talking about independence today. I am not planning to install border points on the M25," Khan told the conference in London.
"But on behalf of all Londoners, I am demanding more autonomy for the capital -- right now," he said, adding: "We can't hang around for the outcome of the negotiations before we give Londoners more control".
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Following the shock referendum result, an online petition urging Khan to declare independence and apply for London to join the European Union independently received 175,000 signatures.
"Londoners -- who voted for a different path than the rest of England -- need more self-determination. We need to control our own destiny," Khan said.
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