As a new electrochemical sensing concept, a self-powered sensor shows a good application prospect in the field of analysis. However, it is still a great challenge to improve the anti-interference capability of sensors through reasonable design. In this study, we investigated the difference between the single photoanode and photocathode self-powered sensor and combined the advantages of these two aspects to fabricate a mediator-free self-powered aptasensor based on the dual-photoelectrode system, which combined the biological events from the photocathode. The biological events occurred at the photocathode could avoid the interference caused by the generated hole oxidation of reducing small molecules in the real sample on the photoanode surface, which was helpful to enhance the anti-interference capability of the sensor. Moreover, due to the sufficient Fermi level differentiation between two photoelectrodes, the redox mediator was not necessary. This could avoid the redox reaction caused by the introduction of extra electron donors or electron acceptors occurring before the photoelectrical behavior, thus improving the accuracy of the sensor. According to the influence of the generated biological conjugate on the external circuit, electron transmission between interfaces, and the obstruction of visible light irradiation, the sensitive and accurate detection of the analytical model was achieved. This work provided a proof-of-concept for the establishment of a mediator-free dual-photoelectrode self-powered sensing platform with high sensitivity and strong anti-interference performance.