Extra emergency accommodation for rough sleepers will open across London tonight, as the weather turns colder.
A Met Office cold weather alert will be in effect in the capital from Wednesday evening until Monday morning.
The alert requires social and healthcare services to target services for high-risk groups of people.
Councils across the capital have also been asked by London's mayor to provide extra shelter for the homeless when the temperature falls below freezing.
Rough sleeping increasing amid cost-of-living crisis
It will be the first time since last winter that the severe weather emergency protocol has been activated.
City Hall said all boroughs in London have committed to a pledge that no one will be asked to leave emergency shelters until a more permanent support plan is in place for them.
Data from the Combined Homelessness and Information Network suggests the number of people either new to rough sleeping or living on the streets in London rose 24% between June and September compared to the same period last year, meaning more than 3,600 people slept on the capital's streets.
Mr Khan has warned that rising bills and housing costs had meant increasing numbers of people were sleeping rough on London's streets.
This week he launched his annual winter rough sleeping fundraising campaign to raise money for several charities helping homeless Londoners.
"London's councils and charities will be working even harder this week to support some of the most vulnerable people in our city. On behalf of all Londoners, I thank them for their tireless efforts," the mayor said.