After three years of bitter dispute, Johnson's deal officially became law after clearing all parliamentary hurdles and receiving royal assent on Friday, reports metro.co.uk.
Earlier on Friday, European Council's president Charles Michel and the European Commission president Ursula von der Leyen signed the document in Brussels before it was transported to London on a Eurostar train.
Later in the day, the Prime Minister uploaded a picture of himself smiling at the camera as he put pen to paper with two Union Jack flags in the background.
Taking to Twitter along with the picture, he wrote: "Today I have signed the Withdrawal Agreement for the UK to leave the EU on January 31st, honouring the democratic mandate of the British people. '
"This signature heralds a new chapter in our nation's history."
Downing Street officials said Johnson marked the document with a Parker fountain pen, as is traditional for ceremonial signings in No 10, reports the BBC.
It was witnessed by EU and Foreign Office officials, including the Prime Minister's Chief Negotiator David Frost, and Downing Street staff.
"The signing 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," Johnson told the media on Friday night.
"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 UK, while building a strong new relationship with the EU as friends and sovereign equals."
The UK will keep a copy of the agreement while the original will return to Brussels, where it will be stored in an archive along with other historic international agreements.
Next week's European Parliament vote is seen as all but a formality, after it was backed by Parliament's constitutional affairs committee on Thursday.
Once the UK leaves the EU, it will enter a transition period where it will follow the bloc's rules and regulations while the terms of the future relationship are worked out.
( With inputs from IANS )