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Biosensor working in a self-powered mode has been widely concerned because it produces a signal when the bias potential is 0 V. However, the self-powered mode is used only when the materials have self-powered properties. Conversion of non-self-powered to self-powered through molecular regulation can solve this problem effectively. Here, we fabricated a self-powered photoelectrochemical mode based on co-regulation of electron acceptors methylene blue (MB) and p-nitrophenol (p-NP). AuNPs@ZnSe nanosheet-modified gold electrode (AuNPs@ZnSeNSs/GE) gave a small photocurrent at 0 V. In the presence of MB and p-NP, AuNPs@ZnSeNSs/GE gave the strongest photocurrent at 0 V. Accordingly, an electron acceptor co-regulated self-powered photoelectrochemical assay was fabricated. As proof-of-concept demonstrations, this assay was applied for prostate cancer circulating tumor nucleic acid biomarker, KLK2 and PCA3, detection combined with in situ recombinase polymerase amplification strategy. This assay generated a strong photocurrent and was sensitive to the variation of KLK2 and PCA3 concentration. The limits of detection were 30 and 32 aM, respectively. We anticipate this electron acceptor co-regulated self-powered photoelectrochemical mode to pave a new way for the development of self-powered sensing.