Most　electrochemical　biosensors　for　microRNA　(miRNA)　detection　need　the　immobilization　of　DNA　on　the　electrode;　first,　the　process　is　tedious　and　the　DNA/RNA　hybridization　occurs　on　the　electrode/solution　interface,　resulting　in　low　efficiency.　In　this　study,　a　homogeneous　electrochemical　biosensor　has　been　proposed　for　sequence-specific　miRNA　in　pancreatic　cancer　cells　and　lung　adenocarcinoma　cells,　which　combines　the　advantages　of　the　simplicity　of　immobilization　free　and　high　efficiency　of　duplex-specific　nuclease　(DSN)　assisted　target　recycling　amplification.　A　DNA　probe　modified　with　a　methylene　blue　group　at　the　3＇　terminal　(eMB)　contains　negative　charges　on　its　backbone,　which　cannot　diffuse　easily　to　the　surface　of　the　negatively　charged　indium　tin　oxide　(ITO)　electrode　because　of　electrostatic　repulsion,　so　a　low　electrochemical　signal　was　detected.　In　the　presence　of　miRNA,　eMB　can　recognize　and　hybridize　with　miRNA　to　form　a　DNA-RNA　heteroduplex.　The　eMB　can　be　hydrolysed　by　DSN,　so　miRNA　is　retained.　miRNA　can　cause　a　new　cycle　of　hybridization-cleavage-releasing,　and　this　recycling　process　generates　numerous　short　MB-labelled　oligonucleotide　fragments　(MB-OFs).　The　MB-OFs　with　less　negative　charges　can　diffuse　easily　to　the　surface　of　the　ITO　electrode,　so　an　enhanced　electrochemical　signal　was　detected.　Under　the　optimal　conditions,　the　differential　pulse　voltammetric　(DPV)　response　of　the　system　had　a　linear　relationship　with　the　logarithm　of　the　target　miRNA　concentration　in　the　range　of　0.1　pM　to　10　nM.　The　proposed　biosensor　has　been　successfully　applied　to　detect　miRNA　in　real　samples.