Abstract: ob＜x＞jective: MicroRNA-21 (miRNA-21) can be used as a tumor marker for early breast cancer screening.This study aims to study and design a Cy5 optical stem loop DNA molecular beacon probe to detect microRNA-21, construct a primers stack isothermal cyclic chain replacement fluorescence quantitative analysis method, and achieve highly sensitive detection of miRNA-21. Methods: Stem loop hDNA probe with Cy5 optical label was designed, and isothermal cyclic chain replacement amplification reaction was activated under the pairing initiation of target mirNA-21. Fluorescence quantitative analysis method was used to optimize the experimental dosage, and sensitive and specific detection of miRNA-21 was performed. This study will prove the reliability of this study as a detection method for miRNA-21 by comparing the detection results with qRT-PCR method. Results: The chain replacement isothermal amplification reaction constructed by Cy5 optical hDNA probe designed in this study could detect miRNA-21 with high sensitivity, and the detection limit was as low as 1fM. MiRNA-21 has good specificity and does not react with other miRNAs to produce fluorescence, which can effectively distinguish single ba＜x＞se mutated microRNA(miRNA). There was no significant difference between the detection results of miRNA-21 by the probe and that of miRNA-21 by gold standard QRT-PCR (P > 0.05), which proved the reliability of the detection results of the probe. Conclusions: The Cy5 optical probe used in this study can achieve highly sensitive detection of miRNA-21 with high specificity, in order to achieve screening and diagnosis of early breast cancer patients