The decision marks the start of a tough campaign among rivals seeking to replace Uribe, who after almost eight years in power became one of the country's most popular presidents for his US-backed campaign against leftist guerrillas.
Monica Villamizar, Al Jazeera's correspondent in Bogota, said: "The main reasons for the referendum being ruled unconstitutional lay in technicalities. The court also cited irregularities over the referendum's financing."
Any successor in Latin America's fourth largest oil producer would be unlikely to diverge far from the president's security policies.
Juan Manuel Santos, a former defence minister, closely associated with Uribe's security successes against Farc leftist guerrillas, led in most opinion polls when Uribe was not included as a candidate.
Sergio Fajardo, an independent praised for his performance as mayor of the major city of Medellin, is also making ground, while another former defence minister and three-time candidate Noemi Sanin has recently gained in the polls behind him.
During Uribe's presidency, Latin America's oldest insurgency has ebbed and foreign investment has flowed steadily into Colombia, a country once considered a byword for a violent, failed state.
A political transition could unnerve the local peso currency and benchmark TES debt markets as investors absorb the change in command, but long-term, most analysts see continuity in Colombian stability.
"While a constitutional court rejection may trigger a knee-jerk negative (peso) reaction, this is likely to be transitory as no major shift in economic, regulatory or security policy is expected," RBC Capital Markets said.