Len McCluskey has vowed never to wear a red triangle armband on the picket line as he likened it to being a Nazi concentration camp prisoner.
The Government's Trade Union Bill could require picket supervisors to wear an armband as part of a wider tightening of the rules to strike.
The controversial legislation would impose new minimum thresholds before strikes would be legal.
Unite leader Mr McCluskey suggested a deal on thresholds could be possible if the Government concedes methods of making it easier to carry out ballots - such as letting them take place online for the first time.
But in a furious party conference speech moving a motion condemning the Bill, Mr McCluskey said: "Let me make one thing clear.
"Whatever the law says, I will be on the picket line when Unite members are on strike.
"And I will not be wearing an armband with a red triangle like the trade union prisoners - that's what the Nazis did, trade unionists in the concentration camps of Dachau.
"We won't be doing that."
Mr McCluskey, who won a huge standing ovation at the end of his speech, told activists in Brighton the legislation is "unnecessary, illiberal and spiteful".
And he said it was driven by "crude class resentment of the Tory right-wing".
The motion was backed by a series of speakers at Labour's annual party conference.
Mr McCluskey said he would seek to mobilise opinion to challenge the legislation.
He added: "We need to seize the arguments of democracy and modernisation, the arguments used against us in the past.
"So I say this to ministers today - if your concern about turnout in strike ballots is genuine, then scrap the archaic and undemocratic reliance on postal ballots and give trade unionists the right to secure, secret, workplace balloting.
"Modernise the voting system and there will no longer be a turnout problem. If ministers agree then we can reach an agreement on thresholds."
Labour members will vote on a series of motions later.