London mayor Boris Johnson visited the proposed site in 2009
Conservative leader David Cameron has said building an airport in the Thames Estuary is not among his party's plans.
Mr Cameron said if elected to government in this year's general election it would not be the policy to construct the four-runway airport.
The plan by London mayor Boris Johnson has been met with strong opposition.
He recently told senior colleagues that building an airport in the Thames Estuary could be "the single biggest... and bravest project we do".
But speaking to BBC South East Today, Mr Cameron said: "It's not our policy.
"I mean we don't want to see the third runway at Heathrow and we can stop that from happening.
"We want to see instead high-speed rail and a proper rail hub at Heathrow, so that all those flights... that serve places that could be met by rail, all of those will be met by rail. That's our policy."
He added: "Boris is a great mayor of London doing an excellent job, but building airports is not his responsibility."
Last year, the proposal was described by the government as "irresponsible and disastrous".
But in a statement on Friday, Mr Johnson's office said it was sure the airport would not be out of the question.
'Nail in coffin'
Mr Johnson's plans for an airport on an artificial island in the Thames estuary have been condemned by Kent County Council (KCC), Medway Council, local business leaders and conservationists.
Paul Carter, KCC leader, said he was "absolutely delighted" following Mr Cameron's comments.
"We've always promoted Manston airport as an opportunity to develop a really good regional hub airport.
"I hope that we can get real traction on that because it will bring economic prosperity to east Kent," he said.
Campaigner George Crozer described it as "another nail in Boris's coffin".
"I don't think that we ever thought that Boris Island was part of Tory policy anyway, so it's really great to hear that," he said.
The opposition Liberal Democrats on the Greater London Authority have now called for the mayor to back down on the idea.
Councillor Caroline Pidgeon said the only thing he had succeeded in doing was to "unite councils in Kent and Essex, environmental groups, and now we hear all three main political parties in Parliament against this mad idea".