"It must be closed down, the danger is that great," Aysun Ozarman, 55, one of the protesters at the rally, said.
The court case comes amid continued tension between the AK Party-led government and advocates of secularism, who include judges, army officers and university professors.
The legal challenge was launched shortly after the AK Party moved to lift a ban on headscarves in universities, an issue which is also being challenged in court.
The AK Party says that it will uphold the separation of state and religion, but argues that a rigid interpretation of secularism could violate personal freedoms.
The demonstrators alleged that the European Union was lending undue support to the government.
Turkey's government is aiming for the country to join the EU but the bloc's popularity has suffered among the Turkish public since membership talks began in 2005.
Jose Manuel Barroso, president of the European Commission, said on Saturday while visiting Istanbul that the Turkish high court must apply European standards in the case against the AK Party.
Ollie Rehn, commissioner for EU enlargement, has said that Turkey's attempt to join the EU could be badly damaged if the AK Party is banned.