A sensitive, label-free and time-resolved luminescent aptasensor to detect proteins was developed based on the DNA-enhanced time-resolved luminescence of Tb(3+) and graphene oxide (GO). We found that the DNA no matter with a G-quadruplex structure or not could greatly enhance the long-lived emission of Tb(3+), and the luminescence of DNA-Tb(3+) could be effectively quenched by GO after the DNA-Tb(3+) was adsorbed onto GO. The target protein combined with an aptamer to form a protein/DNA complex restrained the quenching of DNA-Tb(3+) emission by GO. Thrombin and a 29-mer anti-thrombin aptamer were employed as a model analyte and a recognition element. There is a good linear relationship between the aptamer-Tb(3+) complex luminescence with the thrombin concentrations of 1 to 100 nM with a low detection limit of 0.8 nM. Since the time-resolved luminescence can eliminate the unspecific background fluorescence, the proposed aptasensor has been successfully applied in complicated biological samples for thrombin detection. This novel strategy presents a potential universal method for detection of other molecules.