The MP for Bracknell, who intends to break the whip to vote for motion seizing control of the Commons timetable, crossed the floor of the chamber while the Prime Minister beginning a statement on Brexit.
His defection turns the government into a minority administration, even with the support of 10 DUP MPs.
It comes ahead of crucial votes today and tomorrow that could force Mr Johnson to delay the UK’s exit from the EU and prevent a no-deal scenario.
Dr Lee was among a list of at least 14 Tory MPs who had defied a threat from Downing Street to throw them out of the party in order to vote for measures to block a no-deal Brexit.
In a statement, he said the Conservative Party he joined 27 years ago “Is not the party I am leaving today”.
"This Conservative Government is aggressively pursuing a damaging Brexit in unprincipled ways,” Dr Lee said.
“It is putting lives and livelihoods at risk unnecessarily and it is wantonly endangering the integrity of the United Kingdom.
“More widely, it is undermining our country’s economy, democracy and role in the world. It is using political manipulation, bullying and lies.”
He added that he believed the Lib Dems were “best placed to build the unifying and inspiring political force needed to heal our divisions”.
The defection - the third in recent weeks - puts the Lib Dems on 16 MPs. Party leader Jo Swinson said she was “delighted” to welcome Dr Lee, adding: “He shares our commitment to prevent a disastrous No Deal Brexit, and to stop Brexit altogether.”