Asked if he backed the poll, Mr Johnson said: "If Richmond is going to hold a referendum on this issue, then I would totally support it and I would vote No. It is the wrong way for London.
"We have had elections on this. These issues have been well discussed in all our campaigns. Nobody wants to close Heathrow. But you cannot keep expanding it.
"There is no point in building a new short runway which wouldn’t really do the job because it is only a prelude to further expansion."
Mr Johnson has announced his own inquiry into the future of aviation in the capital, which will report back before the Government-backed commission on the same issue, headed by Sir Howard Davies, former head of the Financial Services Authority. He supports the creation of a new airport, on the Thames Estuary.
The Mayor told the London Assembly: "I will be mobilising and alerting Londoners to the risks of the third runway and what we need to do is to bring the alternative solutions forcibly before public.
"At the moment there is an imbalance, because the third runway at Heathrow is the scheme that has been costed, that has been engineered, that has been evaluated, all the designs are there.
"None of the other options have been properly set out. I think it would be right for the Government to introduce some parity into the argument and make sure that the alternative solutions, that we all know exists, are properly canvassed by the Davis Commission."
Ministers have ruled out the expansion of Heathrow before the next general election, due in 2015, but have refused to make the same commitment for future parliaments.
The Davis Commission is due to report back in late 2015, raising suspicions that it will mandate a third runway.