This　work　presents　a　photocurrent-polarity-switching-based　photoelectrochemical　(PEC)　biosensing　platform　for　ultrasensitive　detection　of　microRNA-21　(miR-21)　through　target-triggered　catalytic　hairpin　assembly　(CHA)　for　modulation　of　methylene　blue　(MB)　and　ferrocene　(Fc)　positional　configurations　using　double-shelled　Cu-doped　ZnS　nanocages　(NCs)-Au　nanoparticles　(NPs)　as　photoactive　materials.　In　the　presence　of　miR-21,　the　assembly　of　MB-labeled　HP1　and　Fc-labeled　HP2　leads　to　the　generation　of　a　large　amount　of　double-stranded　DNA　(HP1-HP2),　which　pushes　MB　away　from　the　electrode　surface　and　brings　Fc　close　to　the　electrode　surface,　resulting　in　effectively　quenching　the　enhanced　PEC　signal　to　activate　the　photocurrent-polarity-switching　system.　Benefiting　from　the　distance-controllable　strategy,　the　designed　PEC　bioanalysis　can　effectively　eliminate　false-positive　and　false-negative　signals　due　to　the　change　of　different　signal　expression　patterns　(from　traditional　the　＂signal-on＂　mode　to　the　photocurrent-polarity-switching　mode),　thereby　significantly　improving　the　sensing　specificity　and　sensitivity.　The　proposed　PEC　sensing　system　exhibited　satisfying　photocurrent　responses　toward　target　miR-21　within　the　working　range　from　1.0　fM　to　1　nM　at　a　low　limit　of　detection　(LOD)　of　0.58　fM.　More　importantly,　we　demonstrated　the　successful　integration　of　the　proposed　PEC　biosensor　with　a　handheld　wireless　device　for　instant　detection　of　miR-21　concentrations　in　practical　samples.　©　2023　American　Chemical　Society.　All　rights　reserved.