The season finale will be played at West Ham United’s 60,000 capacity London Stadium on Sunday, June 5 at 3pm, the National League announced on Monday.
The match will be broadcast live on BT Sport.
Chesterfield are currently in a play-off position with four games remaining and are aiming to return to the Football League for the first time since 2018.
National League chairman Jack Pearce said the venue - which witnessed some historic sporting moments ten years ago during the London 2012 Olympics - ‘is the perfect fit for some more magic’.
National League general manager, Mark Ives, added: “It is a fantastic sporting arena with a rich history and very befitting of our showpiece promotion final.
“Whilst we don’t know who will get to the promotion final yet, what we can now promise is a stadium worthy of hosting such a huge game.”
And Graham Gilmore, CEO of London Stadium, added: “We are proud to be hosting the National League play-off final this year, with the contenders vying for that coveted place in the Football League.
"We have witnessed some truly epic matches at our venue this season and this match will be another one of those special occasions at London Stadium.”
Last year’s final, which Hartlepool United won on penalties after a thriller against Torquay United, was held at Bristol City’s Ashton Gate stadium.
West Ham have played at the London Stadium since 2016 after leaving Upton Park.