Addressing 6,000 people in London, Khan blasted Johnson over the Leave campaign's focus on immigration, while Johnson returned fire over the UK's loss of sovereignty.
The current mayor accused his predecessor of “project hate”, while Johnson said Khan was “completely wrong” over the UK's ability to contradict European courts.
Khan argued that scores of jobs in London are “directly dependent” on the European Union, but the Conservative MP accused him of “running down of our city and country”.
“They underestimate our ability to do better if we are left to do it on our own,” Johnson said.
The two shared running clashes throughout a two hour battle that also saw Scottish Conservatives leader Ruth Davidson make her mark on a national stage.
“The Britain that I love works with its friends and neighbours. It doesn't walk away from them,” Davidson said, delivering closing remarks for the Remain camp.
“'There is nothing more positive than having a stronger economy supporting jobs and opportunities.”
At the same time, energy minister Andrea Leadsom also landed blows for the Brexit camp, arguing that 10,000 elected European elected officials earn more than the Prime Minister.
“That gravy train is continuing,” Leadsom said, drawing one of many cheers from the audience inside the SSE Arena, Wembley.
However, it was Johnson who secured the evening's only standing ovation, saying the campaign had showed a clear choice between fear from the Remain camp, and hope from Leave.
"We say they are woefully underestimating what this country can do," Johnson said. "Thursday can be our independence day."