Transport spokeswoman Theresa Villiers on the plans
The Tories say they would scrap plans for a third runway at Heathrow and build a high-speed rail line instead.
They are proposing to create a new line linking London St Pancras, Birmingham, Manchester and Leeds - saying it would cut Heathrow flights by 66,000 a year.
Transport spokeswoman Theresa Villiers said it had been a hard decision but the party would "not run" from it.
The party says it would cost £1.3bn a year for 12 years. Airport operator BAA said it was offering a "false choice".
Ms Villiers, the shadow transport secretary, told the party's conference in Birmingham: "This is one of the hardest decisions we have faced as a party and we will not run away from it.
"That's why I can announce this morning that a Conservative government would say no to a third runway at Heathrow."
She said rail could offer a viable alternative to short-haul flights and mean about 66,000 fewer flights a year, 30% of the planned capacity of the third runway, eventually rising to 44% with a more extensive high speed rail network in the UK.
PROPOSED HIGH SPEED RAIL
Journey times from London (current: HSR time) To Birmingham: 80mins/45mins To Manchester: 125mins/80mins To Leeds: 125mins/97mins Source: Conservative Party
The proposed 180mph rail link would run between St Pancras in London - the terminus for Eurostar - and Birmingham, Manchester and Leeds. Heathrow, the UK's largest airport, would also be linked in.
It would be a new railway with a new track freeing up the West Coast Mainline for more commuter journeys. Construction would start in 2015, to finish in 2027, should the Conservatives win the next general election.
The party says journeys would be reduced from 125 minutes to 80 minutes from London to Manchester, and from 55 minutes to 17 minutes between Manchester and Leeds.
A Conservative government would spend £15.6bn over 12 years on the project, which they say could be met from within current levels of government capital spend on rail. The private sector would contribute a further £4.4bn.
They would open a competition to build the new rail link and say they will be consulting further with industry and the civil service in the next few months, to ensure the necessary preparations are made before the end of this Parliament.
In her speech Ms Villiers said despite difficult economic times: "This country can no longer put off the decisions necessary to deliver the transport improvements we desperately need ... decisions that Labour have shown themselves so manifestly incapable of taking".
She said high speed rail was a "viable and attractive" alternative to short haul flights and would reduce road congestion, generate economic benefits and improve transport links without the "considerable" environmental penalties of a third runway.
"It will leave a lasting legacy for the future. And it will lay the foundations for a high speed network that I believe will one day stretch across the country," she added.
It is the Tories who cannot face up to the tough decisions needed to support the economy
Earlier she said, a few years ago it would have been "inconceivable" for a Conservative leader to "put the brakes" on Heathrow expansion, which is supported by business leaders and airport operator BAA.
BAA says Heathrow is "jam packed" and needs another runway to compete with other major European airports. A spokesman said Britain needed both a third runway and a high speed rail line.
"To prioritise one over the other is a false choice and will put Britain's future competitiveness at risk. It is not clear how this proposal will resolve the lack of airport capacity, a problem which must urgently be addressed," he said.
He said flights to Manchester and Leeds Bradford airport made up less than 3% of Heathrow's total flights.
Transport Secretary Ruth Kelly said the plans were "politically opportunistic" and would be "hugely damaging" to the national interest.
"Even if every flight from Manchester and Leeds/Bradford to Heathrow were replaced by a new high speed line then Heathrow would still be operating at 97% capacity," she said.
"It is the Tories who cannot face up to the tough decisions needed to support the economy, hiding behind unfunded, ill-thought-through policy announcements which only reinforces their reputation as lightweight, shallow and only interested in grabbing a headline."
A government decision on whether to go ahead with a third runway is expected later this year.
Liberal Democrat transport spokesman Norman Baker said: "It seems the Tories have finally recognised the need for high-speed rail and the scrapping of the third runway at Heathrow, which Liberal Democrats have long called for.
"However, we will have to wait and see the small print before we know if this is just another of David Cameron's empty promises."
He added that the plans were "at odds" with a suggestion from London's Conservative mayor Boris Johnson that a new airport on an artificial island in the Thames estuary could be used to ease overcrowding instead.
Meanwhile, the SNP said the proposals failed to take Scotland into account.
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