The Mayor, who is a Muslim, said: "I don't think he should be coming on a state visit while the ban is in place, I couldn't be clearer."
It comes as a petition to cancel the new President's state visit reaches more than a million signatures.
A government spokesman has said the visit will still go ahead.
Mr Khan has previously openly criticised Trump's policy to ban refugees and citizens from seven countries, saying "it flies in the face of the values of freedom."
He said: "The USA has a proud history of welcoming and resettling refugees.
"The President can't just turn his back on this global crisis - all countries need to play their part.
"While every country has the right to set its own immigration policies, this new policy flies in the face of the values of freedom and tolerance that the USA was built upon."
Although he praised Prime Minister Theresa May for saying the government does not agree with the policy, which will affect many British citizens who have dual nationality, the Mayor does not agree with the visit.
He added: "As a nation that, like the USA, values tolerance, diversity and freedom, we cannot just shrug our shoulders and say: "It's not our problem."
Calls have also been made for Westminster City Council’s new leader and high ranking officials to block the proposed state visit of Donald Trump.
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