Mr Khan has instructed Transport for London to bring forward 10 of its small sites for development under his Small Sites, Small Builders programme.
The sites will each have the capacity for between two and 42 homes, and only SMEs will be allowed to bid for the builds.
The initial 10 plots of land will deliver 111 new homes, 68 per cent of which will be affordable.
Two of the 10 sites – at Cable Street in east London and Christchurch Road in Lambeth – will be 100 per cent affordable housing.
Mr Khan said SMEs will be able to bid for the contracts through a simple bidding process with standardised legal contracts.
Over the past decade the number of houses being built by SMEs in the capital has halved.
The mayor’s team has entered discussions with other public sector landowners about opening up smaller sites, and a review of the 10-site pilot will inform how the programme is extended.
TfL is also forwarding a number of larger sites for housing, with the client set to open up land for 3,000 homes this year.
Mr Khan said: “The number of small sites coming forward has halved in the last decade, and we have lost almost a third of all small and medium-sized homebuilders operating in the capital.
“Through my new small sites programme, I want to make more public land available to help contribute not only to tackling the housing crisis in London, but also to reinvigorating our small and medium-sized homebuilding sector.”
Federation of Master Builders London director Barry Mortimer said: “If we are to build the 66,000 new homes we need each year in London we need to unlock the potential of small and medium sized housebuilders.”