Khan took the step, which allows for closer coordination between different public agencies, after Britain reported on Friday (December 17) the largest 24-hour increase in the number of new cases since the pandemic began.
Hospital admissions and health worker absences are rising, Khan said, "so I've taken the decision, in consultation with our partners, to declare a major incident today."
"This is a statement of how serious things are," he said.
Khan, from the opposition Labour Party, also declared a major incident in January when rising COVID-19 cases threatened to overwhelm hospitals.
The Omicron variant is estimated to account for more than 80% of new COVID-19 cases in London.
Prime Minister Boris Johnson was due to chair an emergency committee meeting over the weekend with the devolved administrations in Scotland, Wales and Northern Ireland, which have their own powers over public health.
Sky News said government ministers were being briefed on the latest coronavirus data on Saturday.
The Times newspaper reported that officials were preparing draft new rules that, if introduced, would ban indoor mixing in England except for work for two weeks after Christmas when pubs and restaurants would be limited to table service outdoors.
People would be able to meet in groups of up to six outdoors, the newspaper said.
But ministers were yet to formally consider the plans, The Times said.
A government spokesperson, asked about The Times report, said the government would continue to "look closely at all the emerging data and we'll keep our measures under review as we learn more about this variant."
Johnson said on Friday "we are not closing things down."