Some 12,000 people from more than 100 pro-secular associations waved Turkish flags as they marched to the mausoleum of Mustafa Kemal Ataturk, the founder of modern Turkey. Many carried posters of Ataturk.
"Turkey is secular and it will remain secular," they chanted during the march which was broadcast live on Turkish TV channels.
Secular Turks have felt increasingly threatened by a rise in Islamism under the AK Party.
There was outrage earlier this year when a high court judge, who had ruled against a teacher for wearing a headscarf to school, was shot by a lawyer. Tensions have been rising since.
Senal Sarihan, the president of the Republican Women's Association, said: "The gains of the republic were being rolled back one by one. Today is the day to rise up for the Republic."
Ahmet Necdet Sezer, the Turkish president, who is a staunch secularist, is due to retire in May 2007. Parliament, which is dominated by legislators from the AK Party, will choose the next president.
Sener Eruygur, the former commander of Turkey's paramilitary forces and president of the Ataturk Thought Association, warned against alleged plans by Recep Tayyip Erdogan, the Turkish prime minister, to run for president in May.
Eruygur said: "There are plans to occupy the presidential palace a symbol of secularism in Turkey."
Erdogan has his eyes on the
Although largely ceremonial, the presidency has become a symbol of secularism under Sezer. A former Constitutional Court judge, Sezer has vetoed a record number of laws that he deemed violated the secular constitution and has blocked government efforts to appoint hundreds of reportedly Islamic-oriented candidates to important civil service positions.
Citing the constitution Sezer said: "The state's ... order cannot be based on religious rules. Religion or religious sentiments ... cannot be used for personal or political gains."
Sezer has recently cautioned against the threat of Islamic fundamentalism.
Erdogan's government denies that it has an Islamic agenda, but pro-secular Turks say that the government is slowly moving the country toward increased religious rule and threatening the secular state.
Since taking power, Erdogan has shown his commitment to EU membership by enacting sweeping reforms that allowed the country to start accession talks in October 2005.
He has also stoked secularist concerns by speaking out against restrictions on wearing Islamic-style headscarfs in government offices and schools while taking steps to bolster religious schools.
Erdogan tried to criminalise adultery before being forced to back down under intense EU pressure. Some party-run municipalities have taken steps to ban alcohol consumption in government tea houses.
The presidential palace is a government office and if Erdogan were to become president, the headscarf worn by his wife, Emine, could cause further tensions.