The Commons Speaker said the motion the Government wanted to bring before MPs on Monday was the same "in substance" as the one considered on Saturday.
He said it would be "repetitive and disorderly" to debate the motion again.
The Prime Minister abandoned Saturday's vote on his Brexit deal when the Commons passed an amendment which withholds approval until the deal has been passed into law. He had hoped to get approval for his Brexit plans on Monday afternoon.
But Mr Bercow said because the circumstances and the substance of today's motion were the same, a vote should not go ahead.
He told MPs: “This matter was decided fewer than 49 hours ago.
"After more than three hours of debate the House voted by 322 to 306 for Sir Oliver Letwin’s amendment, which stated that ‘this House has considered the matter but withholds approval unless and until implementing legislation is passed’.”
Mr Bercow continued: “Today’s motion is in substance the same as Saturday’s motion, and the House has decided the matter.
“Today’s circumstances are in substance the same as Saturday’s circumstances.
“My ruling is therefore that the motion will not be debated today as it would be repetitive and disorderly to do so.”
Mr Bercow cited the so-called "same question convention" preventing the same matter being discussed twice. He called it “a necessary rule to ensure the sensible use of the House’s time, and proper respect for the decisions that it takes”.
The Speaker has previously ruled out repeatedly bringing back votes on the same matter more than once.
He came under fire from some Brexit-backing MPs, including Conservative Sir Bernard Jenkin who said it was "becoming remarkable" how often Mr Bercow pleases "one lot and not the other lot".
Downing Street said the Government was "disappointed" with his ruling, and would now go ahead with the introduction of the Withdrawal Agreement Bill.
"We are disappointed that the Speaker has yet again denied us the chance to deliver on the will of the British people," the Prime Minister's official spokesman said.
The Government is set to publish the Withdrawal Agreement Bill later on Monday, with the second reading debate due to take place on Tuesday.
The Withdrawal Agreement Bill is the legally-binding treaty that must be passed for the UK to leave the EU, while the Government must also win a meaningful vote.
Brexit Secretary Stephen Barclay called on MPs to "respect the referendum" by backing the Bill, warning them: "This is the chance to leave the EU with a deal on October 31."
Ministers insist they could have sufficient support among MPs to get it passed so the UK can depart by the current October 31 deadline.
But, with no Commons majority, Mr Johnson faces a major battle to achieve his pledge to lead the country out of the bloc on that date.