Tuffrey made his announcement on Sunday’s edition of the BBC Politics Show.
Tuffrey told the BBC’s Tim Donovan “the thousands of members across London will decide who they want to be their candidate. I’m hoping they’ll select me.”
The Assembly Member said he would offer “very serious solutions to the problems we face in London.”
Today’s announcement pitches the Assembly Member against former MP Lembit Opik who lost his Montgomeryshire seat in last year’s General Election.
Nominations in the contest to become LibDem candidate close on June 17th with the winner due to be announced on August 12th.
Tuffrey first joined the Assembly in February 2002 when he succeeded Louise Bloom and is the longest serving of the Liberal Democrat’s three serving Assembly Members.
Last year he announced his intention to stand down from the Assembly and had been expected to spend a period away from frontline politics.
However in recent months he has faced calls to enter the race with prominent party members starting a ‘draft Mike Tuffrey’ campaign. Today’s announcement was widely anticipated after weeks of speculation.
Last month a poll of Liberal Democrat members conducted by Liberal Democrat Voice revealed Tuffrey was the choice of 56% of site users.
As a nine-year veteran on the Assembly, Tuffrey has experience in holding both incumbent Mayor Boris Johnson and former Mayor and Labour 2012 candidate Ken Livingstone to account.
His knowledge of City Hall and experience in debating with both potential 2012 rivals is expected to be a major benefit in winning his party’s nomination.
Green Party AM and Mayoral hopeful Jenny Jones said: “The great advantage of an Assembly Member being a mayoral candidate is that they understand the scale and the difficulty of the job. And of course they will know exactly how well or how badly the current Mayor is performing.”
Jones predicted Tuffrey would be “a formidable opponent for the Mayor. He knows where Boris has made quite a lot of messes.”
Mr Livingstone said he welcomed both Opik and Tuffrey to the contest, adding “whatever is happening nationally, LibDems in London have the opportunity to be part of a progressive vision for London.”
VIDEO: Mike Tuffrey speaks to BBC One’s The Politics Show
Confirmed 2012 Mayoral candidates:
Boris Johnson – Conservative
Jenny Jones – Green Party
Ken Livingstone – Labour