A　high　performance　miRNA　biosensor　based　on　effective　click　chemistry　assembly　of　a　Ru(bpy)(3)(2+)　labeled　DNA　probe　and　efficient　electrochemiluminescence　(ECL)　quenching　of　the　Ru(bpy)(3)(2+)/BDEA　(BDEA　=　N-butyldiethanolamine)　system　by　surface-confined　electroactive　methylene　blue　(MB)　dye　is　reported.　When　the　target　miRNA　was　present,　the　ECL　signal　instantly　changed　from　＂light　off＂　to　＂light　on＂　status.　Using　the　specific　miRNA　let-7d　as　the　target　analyte,　this　biosensor　provided　sensitive　detection　over　approximately　six　orders　of　magnitude　(10　fM-10　nM),　with　a　limit　of　detection　of　10　fM　(S/N　=　3).　Detailed　study　of　the　ECL　quenching　behavior　of　the　Ru(bpy)(3)(2+)/BDEA　system　by　MB　in　solution　suggested　that　the　ECL　quenching　involves　a　combination　of　photoluminescence　dynamic　quenching　and　quenching　processes　directly　associated　with　the　redox　reactions,　as　well　as　resonance　energy　transfer.　A　large　binding　constant　of　4.7　x　10(11)　M-1　between　let-7d　and　the　DNA　hairpin　was　estimated　using　an　ECL-based　extended　Langmuir　isotherm　model,　suggesting　remarkably　strong　binding　of　the　target　to　the　probe.　Furthermore,　our　biosensor　exhibited　excellent　specificity　and　reproducibility.　Using　the　developed　system,　the　concentration　of　the　target　miRNA　extracted　from　the　A549　cell　line　could　be　obtained,　demonstrating　the　potential　application　of　the　developed　biosensor　to　practical　biological　sample　analysis.　(C)　2019　Elsevier　B.V.　All　rights　reserved.