A new photoelectrochemical (PEC) biosensor was developed by using carboxyl-functionalized graphene and CdSe nanoparticles. This sensitive interface was then successfully applied to detection of thrombin based on the dual-quenched effect of PEC nanoparticle, which relied on the electron transfer of a bipyridinium relay and energy transfer of AuNPs. After recognition with an aptamer, the PEC nanoparticle was removed and a signal-on PEC biosensor was obtained. Moreover, the bio-barcode technique used in the preparation of PEC nanoparticle could avoid cross-reaction and enhances the sensitivity. Taking advantages of the various methods mentioned above, the sensitivity could be easily enhanced. In addition, in this work we also investigated graphene that was modified with different functional groups and AuNPs of different particle sizes. Under optimal conditions, a detection limit of 5.9×10−15 M was achieved. With its simplicity, selectivity, and sensitivity, this strategy shows great promise for the fabrication of highly efficient PEC biosensors.