The fund is intended to boost teaching and pupil achievement in English, languages and science, technology, engineering and maths (STEM) subjects.
The DfE will provide £20m with the Mayor providing a further £4.25m.
Establishing the fund was a key recommendation of the Mayor’s Education Inquiry.
On Monday the Mayor said: ‘Many schools in London are doing tremendous work, and have high expectations of their students. They are not afraid of teaching the ‘crunchy subjects’ like maths, languages and sciences, or putting their children on a meaty diet of reading the classics or learning grammar. As a result, these children will grow up with the confidence and intellectual muscle to compete with the best talent from around the globe and get into the top jobs and universities.
“I now want this level of ambition in all of our state schools in the capital, and the one key thing we have learned from last year’s Inquiry that supporting our teachers’ professional development is vital to achieving this. The London Schools Excellence Fund is going to turbo charge good ideas, empty out pockets of underachievement, and, in the long term, ensure that outstanding teaching is the norm in all our state schools.”
Education Secretary Michael Gove added: “There is some fantastic practice already evident in some London schools and our funding will allow those successes to be shared across the city, and galvanise the country as a whole.
“I look forward to working with the Mayor – who is incredibly ambitious for London’s children – to provide greater challenge and rigour and raise standards.”
The fund has received the cautious backing of the Labour Group on the London Assembly.
Group leader Len Duvall AM said he and his colleagues “welcome any proposals that will increase the aspiration and educational outcomes for London’s school children.”
However Mr Duvall claimed the Mayor “has a record of launching projects which he then quietly drops or doesn’t deliver well” and said his team “will be keeping a close eye on the delivery of this programme and making sure it has a positive impact on the education of London’s school-children.”
The Inquiry also recommended establishing a London Curriculum reflecting the capital’s specific education needs.
City Hall says work is now underway to develop the curriculum which will “bring new depth and life to subject teaching while helping young Londoners to better understand and engage with their city.”
