A　photocurrent-polarity-switching　photoelectrochemical　(PEC)　biosensor　was　developed　for　the　ultrasensitive　detection　of　tobramycin　(TOB)　through　bipedal　DNA　walker　amplification　with　hemin-induced　photocurrent-polarity-switching　using　a　laser-induced　zinc　oxide/graphene　(ZnO/LIG)　photoelectrode.　Specifically,　the　ZnO/LIG　photoelectrode　was　synthesized　in　situ　by　a　laser　direct　writing　(LDW)　technique.　In　the　presence　of　TOB,　it　reacted　with　HP1　and　HP2　and　the　DNA　walker　response　was　activated　to　form　a　stable　hemin/G-quadruplex.　Furthermore,　hemin　induced　a　polarity　shift　in　the　photocurrent　signal.　The　developed　analytical　platform　exhibited　excellent　photoelectron　transport　performance　of　ZnO/LIG,　the　signal　amplification　effect　of　the　DNA　walker　strategy,　and　the　photocurrent-polarity-switching　ability　of　hemin.　Therefore,　it　demonstrated　satisfying　photocurrent　responses　to　the　target　TOB　within　the　working　range　of　20　nM-1.0　mu　M　at　a　low　detection　limit　of　5.43　nM.　The　PEC　platform　exhibited　good　stability,　reproducibility,　sufficient　sensitivity　and　high　selectivity　for　complex　experimental　samples.　Moreover,　the　photocurrent-polarity-switching　PEC　biosensor　improved　the　anti-interference　ability　and　avoided　false　positives　or　negatives.　A　photocurrent-polarity-switching　photoelectrochemical　(PEC)　biosensor　was　developed　for　the　ultrasensitive　detection　of　tobramycin　(TOB)　through　bipedal　DNA　walker　amplification　using　a　laser-induced　zinc　oxide/graphene　(ZnO/LIG)　photoelectrode.