A　novel　electrochemiluminescence　(ECL)　biosensor　was　developed　for　high　sensitive　and　selective　detection　of　miRNA-21　based　on　the　efficient　and　specific　toehold-mediated　strand　displacement　(TMSD)　amplification　with　Ru(phen)32+　loaded　DNA　nanoclews　(NCs–Ru(phen)32+)　as　signal　tags.　The　stable　DNA　nanoclews,　synthesized　by　a　simple　rolling　circle　amplification　reaction,　were　employed　to　load　with　Ru(phen)32+　efficiently　as　ECL　signal　tags　to　amplify　the　signals.　As　for　TMSD,　the　substrate　strand　(Sub)　was　initially　hybridized　with　P1　and　P2　to　form　DNA　duplex　structures　with　a　toehold　1.　miRNA-21　could　hybridize　with　the　toehold　1　and　trigger　the　TMSD　amplification　with　the　help　of　assist　strand,　releasing　lots　of　P1　stands　from　DNA　duplex　structures.　The　TMSD　technique　realized　the　converting　and　amplification　of　the　single　miRNA-21　input　to　lots　of　output　DNA　(namely　P1)　with　good　selectivity,　simultaneously.　Output　P1　were　designed　to　expand　the　stem-locked　region　of　HP,　which　were　immobilized　on　the　Au　electrodes　firstly.　Subsequently,　the　opened　HP　could　hybridize　with　the　Ru(phen)32+,　capturing　the　ECL　signal　tags　closed　to　the　sensing　surface.　The　ECL　intensity　of　the　system　had　a　linear　relationship　with　the　logarithm　of　the　miRNA-21　concentration　in　the　range　of　1.0　fM　to　100　pM　with　a　limit　of　detection　of　0.65　fM.　The　strategy　was　further　applied　to　detect　miRNA-21　in　complex　samples,　and　the　result　was　consistent　with　the　qRT-PCR.　©　2019　Elsevier　B.V.