Councils across the capital can bid to become the first pilot zone and receive £75,000 towards testing and developing ideas to boost high streets at night, such as running night-time markets, testing longer opening hours, or helping shops and hotels host cultural events. The results of the pilot will be used to shape future plans across the capital.
The announcement comes after research revealed that 92% of councils in England believe that the night-time economy can be key in preventing the decline of high street retail.
The mayor has also announced the funding of a new Safer Sounds Partnership, designed to make it easier for organisers to put on live music events, offer training and support venues to bring consistent licensing practice across the capital.
Both new initiatives have been announced in the mayor's response to a report by his Night Time Commission.
Khan said: "Our new Night Time Enterprise Zone will help to boost high streets by delivering innovative ideas that will draw Londoners to their town centres after 6pm, while the Safer Sounds Partnership will provide support for and drive up standards across the music industry."
Night czar Amy Lamé said: "Through our Night Time Enterprise Zone pilot we will see how innovation can be used to boost our high streets. London's live music scene makes a huge contribution to our capital after dark, and I'm delighted that the Safer Sounds Partnership will enable venues and promoters to work together with police and councils to help London's music scene thrive."
Kate Nicholls, chair of the Night Time Commission, said: "I am really delighted that the mayor has responded so positively to the recommendations set out in our report and listened carefully to the views Londoners gave us about how best to improve the life of the city at night. I'm particularly pleased that the three central recommendations - the 'think night' test, the data-led observatory andNight Time Enterprise Zones - are to be taken forward."
Hannah Wadey, chief executive of the Safer Business Network, said the Safer Sounds Partnership "represents a real commitment" to bringing together the live music industry, the police and local authorities to raise standards.
Jasmine Whitbread, chief executive of London First, said: "London's high streets are struggling against a backdrop of higher business rates and online competition, so this is welcome move that takes the capital a step closer to becoming a truly 24-hour city."
Eddie Curzon, CBI London director, said measures like the Night Time Enterprise Zones "could provide both businesses and Londoners more opportunity to benefit from, and enjoy, the capital's vibrant night time economy".