Photoelectrochemical (PEC) biosensors carried out the whole reaction process in the same solution, which would limit the sensitivity and selectivity of detection in the sensing system. Herein, we reported a promising new cathode-anode spatial division PEC platform based on the two-electrode synergistic enhancement strategy. With the photoanode and photocathode integrated in the same current circuit, the platform exhibited an increased photocurrent response, as well as an improved anti-interference ability led by separating the two electrodes spatially. In this proposal, red light-driven AgInS2 nanoparticles (NPs) served as the photoanode to build biometric steps and amplify the signal, whereas p-type PbS quantum dots were selected as the photocathode to increase the signal. With the participation of alkaline phosphatase (ALP) labeled on Au NPs-DNA, ascorbic acid 2-phosphate was catalyzed to produce ascorbic acid as an electron donor, resulting in the enhancement of the PEC signal. Interestingly, in the presence of miRNA-21 and T7 Exo, the one-step DNA walker amplification can be triggered to reduce the PEC signal by releasing ALP-Au NP-DNA. The constructed PEC biosensor exhibited a detection limit of as low as 3.4 fM for miRNA-21, which was expected to be applied to early clinical diagnosis. Also, we believe that the proposed cathode-anode spatial division PEC platform can open up a new view for the establishment of other types of PEC biosensors.