MicroRNAs (miRNAs) play important roles as significant biomarkers in disease diagnostics. Here, an electrochemical biosensor was developed for the quick, sensitive, and specific detection of miRNAs from human-serum samples using three-dimensional (3D) DNA tetrahedron-structured probes (TSPs) and duplex-specific nuclease (DSN). The designed TSPs were composed of a recognition sequence that corresponded to a target miRNA and a G-quadruplex sequence that was combined with hemin to mimic the biocatalytic functions for H2O2 reduction and l-cysteine oxidation. After hybridization with miRNA, the TSPs were immobilized on the Au electrode to shape the DNA-RNA double strands, which could be discriminated by DSN for hydrolysis of the DNA in the heteroduplexes to generate significant change in the reduction currents. Under optimal conditions, the biosensor showed a wide linear response ranging from 0.1 fM to 0.1 pM, with a low detection limit of 0.04 fM. Meanwhile, the method showed acceptable accuracy and precision for the determination of miRNAs in serum after a series of assessments.