A simple and universal aptamer-based label-free approach for highly selective and sensitive fluorescence detection of thrombin was designed.The method is based on the specific recognition ability of label-free aptamer towards their targets,and PicoGreen dye was used to transduce the fluorescent signal of the double strand DNA duplex.As an asymmetric cyanine dye,PicoGreen reagent does not produce any fluorescence signal when it extsts alone.However,upon binding to dsDNA,it shows a significant fluorescence signal whereas no significant fluorescence change could be observed when it binds to ssDNA.Based on this property,it was used in the detection of thrombin.The results showed that a dynamic range of 1.0×10-14-1.0×10-7 mol/L(r2=0.99) was obtained with a detection limit as low as 1.0×10-14 mol/L.In order to determine the specificity of this method,the sensing system against two interfering substances such as bovine serum albumin(BSA) and cytochrome C(Cyt C) was tested.It is found that the interfering substances(each at 1.0×10-8 mol/L) could not effect on the detection of thrombin,which indicated the high selectivity of the sensing method toward thrombin.The present method was successfully applied in the determination of thrombin in real human serum with average recoveries of 97%-102%.The experiments results demonstrated that the proposed method was simple,specific and sensitive,and it was hopeful to apply in the field of medical clinical diagnosis.