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MicroRNAs (miRNAs) have been identified as important biomarkers with great significance for diagnosis and treatment of various diseases. However, their unique properties, such as small size, high sequence homology, and low abundance, make quantitative analysis of miRNAs extremely challenging. Herein, we reported a cascade catalytic hairpin assembly (CCHA) for sensitive and selective detection of miRNA with three kinds of hairpin probes (HP1, HP2, and HP3). In the presence of target miRNA, a series of toehold-mediated intermolecular DNA strand displacement and hybridization was activated among HP1, HP2, and HP3 to assembly numbers of DNA nanoobjects. During this period, the fluorescence response was greatly intensified to indicate the presence and expression level of interested target miRNA. We have demonstrated that the proposed method exhibits a high assay sensitivity to detect low concentration target and an excellent sequence specificity to distinguish even a single-nucleotide difference in vitro. Moreover, we also demonstrated that our design enables the intracellular imaging of miRNA in live cancer and normal cells. These results showing the promising potential of our CCHA for powerful biosensing, clinic diagnosis, or prognosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)