miRNA has been serving as an ideal biomarker for diagnosis, prognosis, and therapy of many severe diseases. In this study, we have developed an amplified electrochemical method for miRNA detection using T7 exonuclease (exo) and copper nanoparticles (CuNPs). Double-stranded DNA modified on the electrode surface is used as the template for in situ synthesis of CuNPs as excellent electrochemical signal sources. Two cycles of DNA cleavage reactions are carefully designed according to the catalytic activity of T7 exo and occur in the solution and at the electrode surface, respectively. The two cycles are integrated for cascade signal amplification. Briefly, target miRNA triggers the first cycle and its product triggers the second cycle, which destroys the template on the electrode for CuNPs synthesis. As a result, electrochemical signal is decreased and can be used to reflect the level of initial miRNA. Due to T7 exoassisted cascade signal amplification and intense electrochemical responses from CuNPs, the biosensor is developed with excellent sensitivity. A linear range from 10-16 to 10-13 M and the limit of detection as low as 4.5 × 10-17 M are achieved. Meanwhile, it shows the capability of discriminating single base mismatch and exhibits the eligibility in the analysis of miRNA extracted from cells. Therefore, it has great potential for biomedical research and disease management.