It was a final opportunity for the two camps to win over voters, with polls showing a razor-tight race less than 36 hours before a vote that will shape the future of Europe.
Panellists locked horns over immigration, as the pro-EU London Mayor Sadiq Khan tore into his predecessor Boris Johnson, a key campaigner on the "Leave" side.
"You're telling lies and you're scaring people," Khan declared as he brandished a "Leave" leaflet warning that majority-Muslim Turkey could join the EU.
"They say we have no choice but to bow down to Brussels. We say they are woefully underestimating this country and what it can do."
"That's scaremongering, Boris, and you should be ashamed... you are using the ruse of Turkey to scare people to vote Leave," Khan said to cheers from the audience.
Johnson threw the criticism back at Khan, saying the pro-EU side had run a "Project Fear" by warning that leaving the 28-member bloc would damage Britain's economy.
"They say we have no choice but to bow down to Brussels. We say they are woefully underestimating this country and what it can do," Johnson said.
The Conservative lawmaker promised Britain an "independence day" on Thursday if it voted to leave, bringing sections of the audience to their feet in prolonged applause.
The prospect of Britain becoming the first state to defect from the EU in the bloc's 60-year history has raised fears of a domino-effect collapse of the European project.
European Commission president Jean-Claude Juncker earlier Britain against "an act of self-harm" he said would endanger everything Europeans had worked together to achieve.
Pro-European panel members Scottish Conservative leader Ruth Davidson, left, and Mayor of London Sadiq Khan, centre, with TUC General Secretary Frances O'Grady. Source: AAP
'Mist of blood'
As the audience filed into the 12,500-seat Wembley Arena, which often hosts global music stars, they were serenaded by pro-Remain demonstrators singing "All You Need is Love".
Organisers Avaaz said the serenade was an attempt to counter the "fear and division" of the campaign.
But the two sides remained deeply opposed and the audience split among equally vocal "Remain" and "Leave" crowds.
"It felt like a football atmosphere... it felt very hostile. You could tell there was almost a mist of blood in the air," said Michael Flaxington, 21, a student from Kent.
Retiree Linda Mayne, 60, also from Kent, said the debate was well-argued on both sides but had not swayed her from her conviction to vote "Leave".
"I support Leave because I want the UK to have our own democracy back, to be able to control ourselves," Mayne said.
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But 21-year-old student Anton Georgiou said the Leave side's "take back control argument" was "an empty slogan with no detailed plan whatsoever".
As the debate concluded, the Daily Mail newspaper announced it was endorsing Brexit.
"Lies. Greedy elites. Or a great future outside a broken, dying Europe," read its front page. "If you believe in Britain vote Leave."
Two newspapers, the Daily Express and The Sun, carried front-page stories reporting that Queen Elizabeth II was challenging guests to give her "three good reasons" why Britain should stay in the EU.
Earlier this year, Buckingham Palace issued a rare complaint over a previous article by The Sun that claimed the queen favoured Brexit, a challenge to the monarch's long-held position of political neutrality.
The Times, which has backed Britain remaining in the EU, published a warning from hundreds of business leaders, including Virgin boss Richard Branson and US media mogul Michael Bloomberg, warning that Brexit could cause an "economic shock".
Members of the public take part in a 'Remain In' Rally in Trafalgar Square, Central London, Britain, 21 June 2016. Source: AAP
Tight race
Earlier, Prime Minister David Cameron warned that future generations would inherit a damaged, diminished economy if the Leave side prevailed and urged voters to think of their children.
"If we vote out, that is it. It is irreversible. We will leave Europe for good and the next generation will have to live with the consequences," Cameron said.
Global financial markets, which rallied the previous day following opinion poll gains by the "Remain" camp, rose again slightly on Tuesday, despite new surveys showing a tight race.
A poll by Survation gave "Remain" 45 percent and "Leave" 44 percent, with 11 percent undecided.
The websites of six major bookmakers showed the odds heavily pointing to a "Remain" vote, with the likelihood of Britain staying in put at around 80 percent.
The latest surveys were mostly conducted after the brutal murder of Jo Cox, a 41-year-old Labour lawmaker and mother of two, who was shot and stabbed in her northern English constituency on Thursday.
Her alleged killer, 52-year-old Thomas Mair, gave his name as "Death to traitors, freedom for Britain" at his first appearance in court after being charged with her murder.
In an interview with the BBC on Tuesday, Cox's widower Brendan said she had been "worried" the debate may have been "whipping up hatred".
