Selective　and　sensitive　detection　of　microRNA　is　crucial　for　early　diagnosis　and　pathogenesis　of　disease.　Here,　we　established　a　novel　electrochemical　biosensor　for　simple　and　accurate　analysis　of　the　tumor　biomarker　microRNA-141,　which　was　based　on　in-situ　catalytic　hairpin　assembly　(CHA)　actuated　DNA　tetrahedral　(DTN)　interfacial　probes.　Two　hairpin　structures　used　for　CHA　reaction　were　placed　on　the　DTN,　in　which　the　hairpin　H1　on　the　one　vertex　of　DTN　and　hairpin　H2　embedded　in　adjacent　edge,　respective.　The　target　microRNA-141　could　open　the　hairpin　H1　and　activated　the　in-situ　CHA　reaction　between　H1　and　H2　to　alter　the　conformational　of　DTN,　increasing　the　chances　of　the　direct　interaction　between　methylene　blue　(MB)　and　the　electrode　surface,　leading　to　an　increase　in　the　electrochemical　signal.　Meanwhile,　the　released　miRNA-141　could　unfold　another　H1,　enabling　the　enzyme-free　recycling　of　the　target　to　obtain　amplified　electrochemical　signals.　Moreover,　the　in-situ　catalytic　hairpin　assembly　reaction　on　DTN　could　shorten　the　reaction　time　and　enhance　the　sensitivity.　The　established　biosensor　exhibited　a　wide　linear　dynamic　range　of　miRNA-141　from　1　fM　to　100　pM　with　a　detection　limit　of　0.32　fM.　Besides,　the　approach　can　discriminate　the　target　miRNA　from　mismatched　ones　with　excellent　selectivity　and　can　be　successfully　applied　in　diluted　serum　samples,　holding　great　potential　for　sensitive　detection　of　various　biomarkers　clinically.　©　2021　Elsevier　B.V.