Stone had been rumoured for the part of Mary Jane Watson, but Sony Pictures have revealed that the 21-year-old will instead play Peter Parker's college girlfriend Gwen Stacy.
In a statement, director Marc Webb said that Stone's "stunning" chemistry with Spider-Man actor Andrew Garfield made her the clear choice.
"At the heart of the story of Peter Parker is not only the amazing Spider-Man, but also an ordinary teenager who is wondering what he has to do to get the girl," Webb said. "Andrew and Emma will bring everything audiences expect to these roles, but also make them their own."
Columbia Pictures president Matt Tolmach added: "It's been an incredible journey for us to watch Emma's star rise as an actress.
"She is extraordinarily talented and has a very special on-screen spark that is perfect for this role. Given her history with the studio, casting her in one of our most important franchises is a real thrill."
The character of Gwen Stacy, who first appeared in 1965 in The Amazing Spider-Man #31, was played by Bryce Dallas Howard in Spider-Man 3 and is noted for her striking blonde hair.
Webb added: "Much to my surprise, it was fun to find out that our choice for Gwen is also a natural blonde."
The currently untitled Spider-Man reboot is due to be released in 2012.