MicroRNAs (miRNAs) are important biomarkers for a variety of diseases. Here, we developed an enzyme-assisted cyclic amplification strategy for an electrochemical method based on a highly sensitive and target-specific catalytic hairpin assembly (CHA) reaction for trace miRNA detection in serum. The miRNA periodically triggers the hairpin probes (H1, H2) to form a three-way structure of DNA through the CHA reaction, which is accompanied by the release of single-stranded DNA (ssDNA1) and miRNA. ssDNA1 binds to the methylene blue (MB)-labeled signal probe (H3-MB) immobilized on the electrode and is cleaved explicitly under the action of an enzyme (Nt.BbvCI), resulting in part of the MB-containing fragments leaving the electrode surface. At the same time, ssDNA1 is rereleased and reused to initiate a new round of enzyme-assisted cleavage. Integrating multiple signal amplification and electrical signal quenching effects enables this strategy to have a low limit of detection (LOD) of 4.67 fM, which can also be used for miRNA detection in serum samples. Furthermore, this strategy can potentially be used for the clinical analysis of miRNAs.