The court was ruling on an appeal by the Spanish government of Prime Minister Mariano Rajoy, who has said Catalonian independence is "nonsense" and will never happen.
But Acting Catalan President Artur Mas, who ran the Catalan government during years of national economic crisis that saw the independence movement swell, said the government of the wealthy northeastern region would stick to its plan.
"Legally, it is clear that the Catalan parliament's resolution is now annulled," he said in an interview with Cadena Ser radio reported by Reuters on Thursday.
"But politically, it is not, because the will of the parliament cannot be annulled and the will of the parliament reflects the will and the ideas of a significant part of the Catalonian population."
The Constitutional Court ruled that the resolution was unconstitutional and said the Catalan assembly could not establish itself as an independent legal and political power above the constitution.
Mas said he wanted to hold another referendum on secession; an informal vote held last year produced a result of 81 percent in favour, although the turnout was only around 40 percent.
Parties favouring a split from Spain won a majority of seats in the Catalan parliament in September, but fell just short of half the vote.