The social networking website registered 8,868 tweets per second during Beyoncé's performance at the MTV Video Music Awards on Sunday night.
The Twitter surge occurred just after viewers saw Beyoncé rub her belly on stage and Kanye West congratulate her husband Jay-Z from the audience.
The previous all-time record for most tweets per second was achieved when 7,196 people posted messages after Japan won the Women's World Cup in July.
Beyoncé confirmed her pregnancy to reporters before the VMAs got underway, but made a formal announcement on stage by saying: "I want you to feel the love that's growing inside me."
Jay-Z is "ecstatic" over the impending birth of his and Beyoncé's first child, a friend has said.
Former Destiny's Child star Kelly Rowland has predicted that Beyoncé will be an "incredible mother".
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Watch Beyoncé's latest music video for '1+1' below: