Accurate　analysis　of　microRNA　(miRNA)　is　promising　for　elucidation　of　cancer　processes　and　therapeutic　effects.　In　this　study,　we　reported　a　new　target-activated,　light-actuated　three-dimensional　(3D)　DNA　walker　on　gold　nanoparticles　for　sensitive　detection　of　miRNA　using　pyrene-incorporated　DNAzyme　analogues.　In　this　design,　the　target　miRNA　activated　the　3D　DNA　walker　system　to　releases　the　walking　arm.　Then,　under　ultraviolet　light　irradiation,　the　pyrene　DNAzyme　on　the　walking　arm　would　consecutively　cleave　the　disulfide　bonds　of　substrate　strands　and　recover　the　fluorescence　signal,　thus　achieving　the　amplified　miRNA　detection.　The　sophisticated　design　of　the　light-actuated　3D　DNA　walker　was　systematically　investigated.　Furthermore,　this　strategy　could　also　be　employed　for　miRNA　analysis　in　serum　samples　with　satisfactory　reproducibility.　Notably,　the　proposed　light-actuated　3D　DNA　walker-based　technique　eliminated　the　need　of　enzymes,　cofactors,　and　RNA　backbones,　thereby　significantly　improving　the　stability　and　efficiency.　Overall,　the　light-actuated　3D　DNA　walker-based　strategy　enabled　facile,　sensitive,　and　specific　detection　of　miRNA　and　provided　new　perspectives　in　diagnostics.　©　2022　American　Chemical　Society.