Abstract: Objective To design a Cy5 optical stem-loop DNA molecular beacon probe for early detection of a tumor marker microRNA-21 (miRNA-21) for screening breast cancer, and establish an isothermal cyclic strand replacement method of fluorescence quantitative analysis through primer stacking, so as to realize the detection of miRNA-21 in a highly sensitive mode. Methods A stem loop hairpinDNA (hDNA) probe with Cy5 optical label was designed, and the isothermal cyclic chain replacement amplification reaction was activated under the pairing initiation of target miRNA-21. Fluorescence quantitative analysis was used to optimize the experimental dosage, and sensitivity and specificity of miRNA-21 were detected. The related findings were compared with the results by real-time qRT-PCR. Results The isothermal replacement amplification reaction constructed by the Cy5 optical hDNA probe designed in this study detected miRNA-21 in a high sensitive mode, with a detection limit of 1 fmol/L. MiRNA-21 had good specificity, without any reaction with other miRNAs to produce fluorescence; single base mutated microRNA(miRNA) was effectively distinguished. There was no statistical difference between the results of miRNA-21 and gold standard qRT-PCR (P>0.05), which indicated that the detection results of the probe were reliable. Conclusions The Cy5 optical probe designed in this study can detect miRNA-21 in a high sensitive and specific mode, so as to achieve early screening and diagnosis of breast cancer patients.