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• A ratiometric electrochemical biosensor for sensitive miRNA detection was developed. • The combination of Mg2+-dependent DNAzyme and competitive host-guest recognition enhanced detection sensitivity. • Satisfactory monitoring of miRNA in human serum samples was achieved. A novel ratiometric electrochemical biosensor was designed for sensitive miRNA-21 detection by combining Mg2+-dependent DNAzyme-cleavage cycling with competitive host-guest inclusion. The dual ferrocene-labeled hairpin DNA (2Fc-DNA)-assembled triple-helix magnetic probe (THMP) and methylene blue (MB)-immobilized reduced graphene oxide/β-cyclodextrin nanocomposites-modified glassy carbon electrode (RGO/β-CD/GCE) were served as target recognition and signal transformation units, respectively. Upon the hybridization reaction between target miRNA-21 and two auxiliary probes, the active DNAzyme was liberated from the caged structure, which bound with 2Fc-DNA to catalyze its cleavage in the presence of Mg2+, and triggered continuous cleavage reaction to obtain a large number of Fc-labeled DNA fragments. After magnetic separation, Fc-labeled DNA fragments could replace MB by the competitive host-guest recognition reaction and accumulate on the electrode surface, resulting in the gradually enhanced Fc and weakened MB signals, respectively. Under the optimal conditions, the constructed biosensor with ratiometric detection mode showed wide concentration range (10 fM−0.1 nM), low detection limit (1.8 fM), high sensitivity, and good selectivity, as well as satisfactory reproducibility and stability. And the developed sensing strategy may provide a new pathway in molecular diagnosis and bioanalysis. [ABSTRACT FROM AUTHOR]