Catalan MEPs defied furious demands to stop waving the flag as they stood in parliament in Strasbourg and took photos.
A panicked Brussels warned Catalonia on Thursday it risks “abandoning the European Union” by splitting from Spain.
Spanish MEP Carlos Iturgaiz, a member of the European People’s Party group, demanded the independence backers “remove the flag of the coup”.
He said: “I’m asking you to remove the flag of the coup, shown by people against a member state of the EU which is Spain.”
After this did not work, he signalled desperately to the European Parliament president Antonio Tajani to intervene.
Mr Tajani said: “Ok, you’ve shown the flag. Now I’m asking you to take your seats we have a serious debate.”
Catalan MEP Ramon Tremosa, from the Alliance of Liberals and Democrats for Europe, who supports the region’s independence from Spain, handed out roses to fellow MEPs and Mr Tajani at the beginning of the session.
The EU is scrambling to avoid a Catalexit, which would see the bloc disintegrate further after an already-chaotic 18 months.