We demonstrate for the first time the development of a mimic peroxidase- and bismuth sulfide (Bi2S3) nanorod-based photoelectrochemical (PEC) biosensor for signal-on detection of polynucleotide kinase (PNK) on the basis of manganese-based mimic enzyme (MnME) catalytic precipitation. We use the hybrid film which consists of Bi2S3 nanorods and Au nanoparticles (AuNPs) as the immobilization matrix of capture probe. The capture probe on the modified electrode can specifically hybridize with the MnME@AuNPs-labeled signal probe to form the double-stranded DNA (dsDNA), generating a PEC biosensor. In the absence of PNK, MnME may stimulate the mimic enzyme catalytic precipitation onto the electrode surface, blocking the interfacial electron transfer and eventually leading to a low PEC signal. While in the presence of PNK, the dsDNA is phosphorylated and subsequently cleaved by lambda exonuclease to release the MnME@AuNPs conjugates from the electrode, leading to the decrease of catalytic precipitation on the surface of electrode and consequently the production of a high PEC signal. Notably, the MnME can be easily synthesized and possesses higher catalytic activity than the manganese-based mimic enzyme. This signal-on PEC biosensor exhibits high sensitivity with a detection limit of 1.27 × 10-5 U mL-1 and an extrembly large dynamic range of 5 orders of magnitude. Moreover, it can be applied for the screening of PNK inhibitors and accurate quantification of PNK activity in cancer cell extracts.