Eliminating false positives or negatives in analysis has been a challenge. Herein, a phenomenon of polarity-switching photocurrent of AgInS2 quantum dot (QD)–DNA nanowires reversing nitrogen-doped porous carbon–ZnO (NPC–ZnO) nanopolyhedra was found for the first time, and a versatile photoelectrochemical (PEC) biosensor with a reversed signal was innovatively proposed for dual-target detection. NPC–ZnO is a photoactive material with excellent PEC properties, while AgInS2 QDs as a photosensitive material match NPC–ZnO in the energy level, which not only promotes the transfer of photogenerated carriers but also switches the direction of PEC current. Furthermore, in order to prevent spontaneous agglomeration of AgInS2 (AIS) QDs and improve its utilization rate, a new multiple-branched DNA nanowire was specially designed to assemble AgInS2 QDs for constructing amplified signal probes, which not only greatly increased the load of AgInS2 QDs but also further enhanced the photoelectric signal. When the target Hg2+-induced cyclic amplification process generated abundant RDNA, the DNA nanowire signal probe with plenty of QDs was linked to the NPC–ZnO/electrode by RDNA, generating greatly amplified polarity-reversed photocurrent for signal “ON” detection of Hg2+. After specific binding of the target (aflatoxin B1, AFB1) to its aptamer, the signal probes of AIS QD-DNA nanowires were released, realizing signal “OFF” assay of AFB1. Thus, the proposed new PEC biosensor provides a versatile method for detection of dual targets and also effectively avoids both false positive and negative phenomena in the assay process, which has great practical application potential in both environmental and food analysis.

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