MicroRNAs (miRNAs) participate in various biological processes during the course of life. The levels of miRNAs can be useful biomarkers for cellular events or cancer diagnosis, thus sensitive and accurate analysis of miRNA expression is crucial for better understanding its functions and the early diagnosis of human disease. Here, we developed a multiple amplification detection method for miRNA based on the host–guest interaction between β-cyclodextrin polymer and pyrene, which takes advantage of the polymerase-aided strand displacement amplification and λ exonuclease-assisted cyclic enzymatic amplification. The proposed method allowed quantitative detection of miRNA-21 in a dynamic range of 1 pM to 5 nM with a detection limit of 0.3 pM and demonstrated good ability to discriminate the target sequence from the single-base mismatched miRNA sequence. Moreover, the assay was applied successfully in a complex biological matrix. We believe that this proposed sensitive and specific assay has great potential as a quantification method for miRNA detection in biomedical research and clinical diagnosis.