The Prime Minister said the UK can now "move forward as one country" after he put his signature to the document in Downing Street on Friday afternoon.
Earlier in the day, the presidents of the European Council, Charles Michel, and the European Commission, Ursula von der Leyen, formally signed the agreement in Brussels.
The Queen gave royal assent to the legislation on Thursday after the legislation cleared both Houses of Parliament.
Mr Johnson said: "The signing of the Withdrawal Agreement is a fantastic moment, which finally delivers the result of the 2016 referendum and brings to an end far too many years of argument and division.
"We can now move forward as one country - with a Government focused upon delivering better public services, greater opportunity and unleashing the potential of every corner of our brilliant United Kingdom, while building a strong new relationship with the EU as friends and sovereign equals."
The PM signed the document with a Parker fountain pen in an anteroom outside the Cabinet Room in Number 10, witnessed by EU and Foreign Office officials who transported the treaty from Brussels.
The European Parliament is now expected to vote to approve the agreement on Wednesday, paving the way for the UK to leave next Friday, January 31.
It will mark the start of an 11-month transition period, during which the UK will continue to follow EU rules, before the final break with Brussels at the end of the year.
Mr Johnson has said he wants to negotiate a comprehensive free trade deal with the EU by the end of the year.
However Mrs von der Leyen and other senior EU figures have warned the Government's stated intention to end the UK's alignment with EU regulations means it will be impossible to achieve within such a tight timetable.
In February, Britain and the EU will begin negotiations on their future ties, racing to strike new relationships for trade, security and a host of other areas by the end of 2020.
Mr Johnson insists he won't agree to any delays in those talks beyond the end of the year. The UK is also keen to begin talks on a free trade deal with the United States, and intends to negotiate simultaneously with the EU and the US.
Speaking at the World Economic Forum in the Swiss ski resort of Davos on Thursday, US Treasury Secretary Steven Mnuchin said a deal with Britain was "an absolute priority of President Trump and we expect to complete that within this year."
US Commerce Secretary Wilbur Ross said a trade deal between the two countries shouldn't be too difficult because their economies are similar.
"Both are very heavily services oriented and within services, such as financial services, there's already a pretty high degree of integration and coordination, so it should be much easier mechanically," he said in Davos.
Ross sought to dampen concerns that a trade deal would mean higher drug prices in the U.K. as U.S. pharmaceutical companies look to gain concessions from Britain's state-run National Health Service.
"What we think is that drugs should have similar prices wherever they are but I don't believe we are in any position to tell the U.K. what they should pay for drugs," he said.