The film, tentatively titled The Newest Hottest Spike Lee Joint, is about "human beings who are addicted to blood. Funny, sexy and bloody".
More than 28 backers each paid $10,000 (£6,300) to sit next to Lee courtside at a New York Knicks basketball game.
Self-described "indie filmmaker" Lee said: "I put my own money in my films."
Writing on the site, he continued: "Indie filmmakers are always in search of financing because their work, their vision sometimes does not coincide with studio pictures.
"I self-financed Red Hook Summer. My fee for Malcolm X was put back into the budget. The truth is I've been doing Kickstarter before there was Kickstarter."
Lee pointed to the recent successes on Kickstarter, such as Scrubs actor Zach Braff raising more than $3m (£1.9m) to direct his next film, and fans of the cancelled TV show Veronica Mars who raised more than $5m (£3.1m) for a film based on the series.
Lee acknowledged critics who have questioned the ethics of successful directors asking fans for money and raising the potential for hurting young filmmakers who struggle to find funding outside of the Hollywood system.
He defended his campaign by saying: "This is another fallacy. The fact of the matter is I'm bringing exposure to Kickstarter, backers to Kickstarter who have never even heard of Kickstarter before."
Lee also defended his decision not to disclose much about the project. He said: "It's a thriller. In order for a film of this type to work the less details the better. For this film to work with the audience, they can't know a whole lot before they sit down in a theatre to see it.
"What I can tell you is this. I have never made a film like this and it excites me very much. I'm doing a semi-genre film about addiction. These people are addicted to blood. However, they are not vampires. It's going to be sexy, humorous and bloody. To me that's a unique combination."
Lee's latest film, a remake of the Korean film Oldboy, starring Josh Brolin, is due to be released in the UK in December.