MicroRNA (miRNA) serves as an ideal biomarker for diagnosis, prognosis, and therapy of various human cancers. The rationally designed three-dimensional (3D) DNA nanomachine was constructed on the matrixes of magnetic beads, and the high density of gold nanoparticles (AuNPs) on each magnetic bead and further enlargement of the AuNPs lead to the anchoring of numerous DNA walkers and signal probes on the AuNPs. With the combination of toehold-mediated strand displacement reaction (SDR), amplified electrochemical detection of miRNA is performed. The existence of miRNA triggers the toehold-mediated SDR and the released DNA walker probe is hybridized with the ferrocene (Fc)-tagged signal probe. The cleavage of the duplex by the nicking endonuclease detaches the signal probe from the magnetic nanocomposites. The oxidation current of Fc moieties was found to be inversely proportional to the concentrations of miRNA-182 between 1.0 fM and 2 pM. The assay is highly selective for discrimination of miRNAs with similar sequences. The feasibility of the method for sensitive detection of the expression levels of miRNA-182 from serum samples of glioma patients at different stages was demonstrated. The sensing protocol holds great promise for early diagnosis and prognosis of the cancer cases with abnormal miRNA expression.