Building flexible and reliable photoelectrochemical (PEC) sensors capable of measuring multiple miRNAs simultaneously still suffers from a challenge. In this protocol, a unique multiplexed PEC biosensor for miRNA-141 and miRNA-155 was developed by Fe₃O₄ @SiO₂ @CdS-HP1-Ag₂S NPs and CdTe QDs-HP2-Au NPs nanobioprobes polarity switching mode. HP1 and HP2 can be simultaneously unfolded by the two targets. The corresponding rigid DNA double-strand structure makes the distance between CdS shells and Ag₂S NPs even greater, exposing more photoactive sites of CdS shells and thus the enhanced photoanode current. Similarly, the exciton-plasmon interaction (EPI) between the CdTe QDs and the Au NPs is disrupted, resulting in the photocurrent recovery of the CdTe QDs. Through external magnetic force, the one-step electrode modification of each above nanobioprobe was realized and the photocurrent polarity switching was also achieved. The linearity range of the designed multiplexed PEC biosensor was from 50 fM to 10 nM, and the detection limits reached 40.8 fM and 37.5 fM, respectively, without using any bioamplification technology. In addition, the manufactured PEC biosensor has significant advantages in compactness, short signal acquisition time, cost-effectiveness, as well as stability and replicability. The unique design provides a new perspective for monitoring multiple miRNAs simultaneously.