In arguments, lawyers for the President maintained that his Twitter account - @realdonaldtrump - is a private one, and is not controlled by the government.
The judges did not agree.
"We conclude that the evidence of the official nature of the Account is overwhelming. We also conclude that once the President has chosen a platform and opened up its interactive space to millions of users and participants, he may not selectively exclude those whose views he disagrees with," the panel concluded.
The case was brought by the Knight First Amendment Institute at Columbia University, which sued on behalf of seven people blocked by Mr. Trump.
Twitter says President Trump has nearly 62 million followers on the social media platform which is his favorite outlet.
Two of the three judges on this panel were appointed by President George W. Bush, the third by President Barack Obama.