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MicroRNAs (miRNAs) play vital regulatory roles in cancer development and a variety of diseases, which make them become promising biomarkers. Here, a simple electrochemical biosensor was developed for highly sensitive and specific detection of target miRNA using mismatched catalytic hairpin assembly (CHA). The target miRNA triggered the toehold strand displacement assembly of two hairpin substrates, which led to the cyclic reuse of the target miRNA and the CHA products. Compared with the traditional CHA, mismatched CHA could decrease the nonspecific CHA products, which reduced the background signal significantly. Under the optimal experimental conditions and using differential pulse voltammetry, the established biosensor could detect target miRNA down to 0.6 pM (S/N=3) with a linear range from 1 pM to 25 nM, and discriminate target miRNA from mismatched miRNA with a high selectivity. It was also applied to the determination of miRNA spiked into human total RNA samples. Thus, this biosensing strategy might become a potential alternative tool for detection of miRNA in biomedical research and early clinical diagnosis.