We proposed a simple and sensitive strategy for the detection of microRNAs (miRNAs) by converting homogeneous assay into surface-tethered electrochemical analysis. Specifically, the biotinylated detection probes (biotin-DNA-biotin) can trigger the in-situ assembly of tetrameric streptavidin (SA) proteins on an electrode surface via the SA-biotin interactions. The (SA-biotin-DNA-biotin) assemblies electrically insulated the electrode interface, thereby blocking the electron transfer of [Fe(CN)]. When the probe was hybridized with the target miRNA, it would be cleaved into small fragments (denoted as biotin-DNA) by duplex-specific nuclease (DSN). The released target miRNA can enter into the next hybridization-enzymolysis cycle, thus leading to the generation of considerable amounts of biotin-DNA fragments. The released biotin-DNA competed with the detection probe to bind SA, thus limiting the in-situ formation of (SA-biotin-DNA-biotin) assemblies. The surface-tethered electrochemical analysis by the dual signal amplification of DSN and (SA-biotin-DNA-biotin) assemblies has been used for the determination of miRNAs in cell lysate with a satisfactory result. The method showed a detection limit down to 10 aM. The "one-step" immobilization-free strategy can be used to design novel biosensors for the detection of other biomarkers.