Herein, by utilizing bipedal DNA walker as booster to adjust the distance of quencher ferrocene (Fc) and sensitizer methylene blue (MB) to photoactive material perylene-3,4,9,10-tetracarboxylic acid (PTCA), a novel "on-off-super on" photoelectrochemical (PEC) biosensor was proposed for ultrasensitive detection of thrombin (TB). Firstly, the PTCA matrix on electrode could provide a high initial PEC signal assisted by depAu. Upon the Fc labeled on hairpin DNA 1 (H-Fc) proximate to PTCA, the PEC signal could obviously decrease to reduce the background signal. Interestingly, the target TB-related bipedal DNA walker implemented the opening of H-Fc for the departure of Fc toward PTCA, which achieved the recovery of PEC signal and exposed the prelocked toehold domain for the hybridization with hairpin DNA 2 labeled with MB (H-MB), thereby making the MB approach to PTCA for achieving the "super on" signal. As a result, this proposed strategy showed a wide linear range from 0.5 fM to 100 nM with a low detection limit down to 0.17 fM for TB detection, providing an efficient and available avenue for sensitive detection of biomolecules in bioanalysis and disease diagnosis.