One youngster held up a sign which read: “Stop the protocol to save the peace!”
A group of women carried a banner bearing the faces of Mr Johnson, Simon Coveney, Joe Biden and Maros Sefcovic.
It read: “Political leaders are not listening!”
Michael Palmer, a councillor for the Ulster Unionist Party (UUP) tweeted his support for the protest which violated Northern Ireland’s Covid regulations.
He urged Boris Johnson’s Government and policymakers in Brussels to “wake up” to the rising sentiment among Unionist communities in the Six Counties.
He wrote: “Massive crowds protesting against the Northern Ireland Protocol in Portadown today confirm there is widespread opposition to it.
“This debunks the myth that it’s a niche issue when it is not.
“It is causing instability in our society. When will the UK Government and EU wake up?”
“You promised you would never allow a border in the Irish Sea.
“You swore it would only happen over your dead body, but when it mattered, you stepped over dead bodies, those Ulstermen and women who sacrificed their lives for this country.”
Mr Holmes added: “The message is loud and clear. The Protocol must go.”
Many Unionists praised the protesters on social media, with one warning: “This isn’t going away until the Protocol does.”
As the protesters made their way through the streets of the town, police officers held a sign informing protesters they were taking part in an illegal protest and that they are liable to arrest.
Under Covid-19 guidelines, gatherings are limited to 500 people outside.
Unionists and loyalists who opposed the protocol say it created a “border in the Irish Sea”, distancing Northern Ireland from the rest of the UK.
Under the Withdrawal Agreement, Northern Ireland continues to follow some EU Single Market rules.
Checks are carried out on goods entering the Six Counties from Great Britain, which Brussels says are necessary in case they end up in the Republic.