Mr Johnson said he feared there was a "stealthy U-turn" under way and vowed to fight the party leadership if it backed a third runway.
He told BBC Radio 4's World at One:
"What I worry about is that we are now seeing a stealthy U-turn being carried out which I don't think is in the interests of London or indeed of the country as a whole because in the end you can expand Heathrow and you can put in a runway...
"Actually it will be a short runway but you have to come back in 10 years time and do another."
Asked if he would campaign against Heathrow expansion he said: "You bet I will, yes."
He said the possibility of a third runway was "being left open" and claimed the commission would not resolve the issue.
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Before the final declaration on Friday, the Conservative candidate looked set to lose the City Hall race to Labour's Sadiq Khan.
The Tooting MP has been the frontrunner in the counting process ahead of Tory challenger Zac Goldsmith.
It's not just London where elections have been taking place today. There were council elections taking place across the home counties.