In　the　developed assay,　multiorbital　3D　DNA　walker　(MO　DNA　walker)　was　applied　as　signal　amplified　protocol　for　enhancing　the　detection　signal　of　the　photothermal　biosensor,　which　was　designed　for　sensitive　detection　of　miRNA　based　on　the　H2S　triggered　conversation　of　photothermal　reagent.　When　the　target　molecule　is　present,　the　DNA　walking　strand　was　released　and　then　hybridize　with　track　strands.　The　landing　of　walking　particles　(WPT)　on　the　tracking　particles　(TPT)　promotes　the　relative　movement　of　the　WPT　around　TPT,　thus　releasing　large　amount　of　horseradish　peroxidase　(HRP)　with　the　aid　of　DNAzyme.　After　reacting　with　Na2S2O3　and　H2O2,　multiple　H2S　can　be　generated　in　situ　based　on　the　catalytic　ability　of　HRP.　Meanwhile,　cubic　Prussian　blue　(CPB)　was　synthesized　and　exhibited　superior　photothermal　response,　which　can　be　served　as　efficient　photothermal　reagent　and　H2S　responsive　acceptor.　Significantly,　the　photothermal　signal　of　CPB　could　be　obviously　reduced　after　challenged　with　H2S　ascribed　to　synchronous　reaction　between　the　ferric　ion　(Fe3+)　and　H2S.　The　improved　walking　area　and　freedom　enable　significant　signal　amplification,　enhancing　the　biosensor’s　performance.　Under　ideal　circumstances,　the　proposed　photothermal　assay　demonstrated　excellent　performance　for　determination　of　miRNA-21.　Graphical　Abstract:　[Figure　not　available:　see　fulltext.]　©　2023,　The　Author(s),　under　exclusive　licence　to　Springer-Verlag　GmbH　Austria,　part　of　Springer　Nature.