Photochemical (PEC) sensors were severely limited for multiplex detection applications due to the cross interference between multiplex signals at the single recognition interface. In this work, a distance-regulated PEC sensor was developed for multiplex detection by using an i-Motif sequence with conformational transformation activity as the signal transduction unit. Through dynamic regulation of the spatial distance between the end site of the functional sequence and the electrode material, the photogenerated electrons on the surface of the sensor were directionally transferred. Thus, a PEC sensor with "signal-on" and "signal-off" dual signal output modes was developed for simultaneous detection of multitarget molecules. Combining isothermal nucleic acid amplification, the PEC sensor constructed in this work was successfully applied to the detection of two virus (Norovirus and Rotavirus) nucleic acid sequences. Under the optimal condition, this bioassay protocol exhibits a linear range of 0.01-100 nM for both viruses with detection limits of 0.72 and 0.53 pM, respectively. In this study, a stimulus-mediated distance regulation strategy successfully addressed the transduction of multiplex detection signals at the single recognition interface of the PEC sensor. It is expected that the technical barriers to multiplex detection of PEC sensors will be overcome and the application of PEC sensing technology will be expanded in the field of environmental analysis.