DNA amplification strategy has been a valuable tool for improving the sensitivity of biosensors. However, the freely diffusing reactants in most DNA amplification strategies limit the rate of DNA reaction, which further affects the amplification efficiency with unsatisfactory sensitivity. In the present work, a novel localized DNA cascade reaction (LDCR) in a DNA nanomachine was designed for high-efficiency target conversion to construct an electrochemiluminescence (ECL) biosensor for ultrasensitive microRNA-21 detection. The DNA nanomachine was constructed by using three-footholds DNA scaffold to immobilize two metastable hairpins and reporter probe and confine them in a localized space. In the presence of microRNA-21, it initiated the LDCR and produced large amounts of mimic target (ferrocene labeled DNA, Fc-DNA) due to the locality effect. Thus, sensitive detection of microRNA-21 could be realized since Fc could effectively quench the ECL intensity of graphitic carbon nitride nanosheets (CNNS) due to the energy and electron transfer from the excited state of CNNS to oxidized species of Fc. Moreover, compared with the other two developed DNA cascade reactions with freely diffusing reactants, the proposed LDCR benefits by shortening the reaction time and improving the amplification efficiency with enhanced sensitivity of the biosensor. Therefore, the proposed LDCR could be used as a highly efficient amplification strategy for ultrasensitive determination of biomarkers with low abundance, which may promote the diagnostic efficiency of disease.