A novel ratiometric electrochemical biosensor for sensitive detection of microRNA was developed by using walker amplification technique. Ag nanoparticles (NPs) and methylene blue (MB) were used as signal probe and as reference, respectively. Firstly, the present target (miR-155) hybridized with DNA2 to release walker (DNA1), and then the walker hybridized with DNA3 to form activated DNA enzyme, inducing cycling shearing of DNA3 in the presence of Mn²⁺, thus a large number of product chains (S1) were produced. Then, S1 opened hairpin DNA (HP1) labeled with MB on the electrode, initiating enzyme-assisted cyclic clipping to generate abundant capture DNA. Thus numerous signal probes (DNA HP2-Ag NPs) were linked for electrochemical detection of target. As the electrochemical signal of MB at capture DNA was constant, so it was used as internal reference. Thus the signal ratio of Ag NPs to MB was used for detection, which can greatly reduce the background. The relation between the I_Ag/I_MB and the concentration of target was 10⁻¹³-10⁻⁶ M, showing wide linear detection range. The biosensor displayed simple design, excellent sensitivity and high specificity for microRNA, showing enormous application prospect in bioanalysis and disease diagnosis fields.