Johnson has sided with environmental groups, such as the Campaign for Better Transport, by claiming that the bridge "in its current form" would be bad for the environment, create traffic congestion and damage efforts to boost public transport in the area.
"Any scheme will have to deal with the issues on both sides of the river in terms of traffic management, safeguarding the environment and public transport usage," said Johnson.
"The current scheme for a Thames Gateway Bridge does not currently fulfil these criteria; therefore I do not support the scheme in its present form."
The controversial proposal to build a road bridge between Beckton and Thamesmead in East London has been hailed as a vital project to boost regeneration in a run down part of the capital.
Transport for London wants to build the crossing but last July a planning inspector said that more evidence was needed to show what regeneration benefits the bridge would bring.
A London First spokesman said that traffic in the area would be kept under control because the bridge would be tolled. It added that without the bridge many vehicles such as delivery vans would have to make longer trips to get across the river, increasing pollution as a result.
"Without the bridge, the regeneration of the area would be stifled," said a London First spokesman who challenged Johnson to publish details of any alternative crossing.
Campaign for Better Transport spokesman said: "We welcome the fact that Boris Johnson is opposed to the current proposals."