Angela Eagle drew laughter from the audience when she accidentally told the debate: "We're not in the European Union."
Boris Johnson was heard saying "fantastic news" following the slip.
Ms Eagle had meant to say Britain was not in the eurozone.
Campaigners for remaining in and leaving the EU have clashed in a fiery debate on ITV.
On a night of fierce debate between Remain and Leave campaigners during the ITV referendum debate, which of the claims actually stacked up?
Analysis of ITV's EU referendum debate.