The Prime Minister also said allowing Russia to win would be a “green light to autocrats everywhere” during his speech.
He made the comments as the Conservative party spring conference in Blackpool on Saturday.
Mr Johnson described the Mr Putin’s actions in Ukraine as “a vicious and a barbarian attack on innocent civilians, the likes of which we haven’t seen since the 1940s”.
He added that if Russia were to win its conflict in Ukraine then this would be the “extinction of any hope of freedom” in Moldova and Georgia.
Mr Johnson said: “I know there are some around the world, even in some western governments … who say that we’re better off making accommodations with tyranny. I believe they are profoundly wrong and to try to renormalise relations with Putin after this, as we did in 2014, would be to make exactly the same mistake.”
He faced backlash after comparing the struggle of the Ukrainian people for freedom to people voting for Brexit.
He said: “I know it’s also the instinct of the people of this country to choose freedom every time … When the British people voted for Brexit in such large numbers … it was because they wanted to be free to do things differently, for this country to be able to do things differently and run itself.”
Mr Johnson has been criticised for his comments, including by Tory peer Lord Barwell, who pointed out Ukraine is seeking to join the EU.
He said: “Apart from the bit where voting in a free and fair referendum isn’t in any way comparable with risking your life to defend your country against invasion + the awkward fact the Ukrainians are fighting for the freedom to join the EU, this comparison is bang on”.
The Ukrainian ambassador to the UK was in the audience while Mr Johnson spoke, alongside the foreign secretary, Liz Truss, and defence secretary, Ben Wallace.
Mr Johnson thanked Wallace for making him read “Putin’s crazy essay” about his invasion plan some months ago.
The Prime Minister said the Russian leader invaded Ukraine because he was afraid of having a democratic neighbour with a free press and free elections.
“You have to ask yourself why he did it – why did he decide to invade this totally innocent country?” he said.
“He didn’t really believe that Ukraine was going to join Nato any time soon, he knew perfectly well there was no plan to put missiles on Ukrainian soil.
“He didn’t really believe the semi-mystical guff he wrote about the origins of the Russian people … Nostradamus meets Russian Wikipedia. That wasn’t what it was about.
“I think he was frightened of Ukraine for an entirely different reason. He was frightened of Ukraine because in Ukraine they have a free press and in Ukraine they have free elections.”