Objective To study the differential expression of miR-21-3p in the peripheral blood of breast cancer patients and patients with benign breast diseases, and explore its diagnostic value and clinical significance in breast cancer. Methods Serum samples from 90 patients with BC and 65 patients with benign breast diseases were collected, and the expression of miR-21-3p was detected by real-time quantitative PCR (qRT-PCR), and the value of miR-21-3p in the diagnosis of BC was evaluated by ROC curve and AUC. The clinical data were further analyzed to explore the correlation between miR-21-3p and different clinical indicators, and pathway analysis was carried out to explore its potential biological functions related to the occurrence of BC.Results The expression level of miR-21-3p in the serum of BC patients was significantly higher than that in the benign control group. The results of ROC curve analysis showed that miR-21-3p showed excellent diagnostic potential with an AUC of 0.96. The expression level of miR-21-3p was correlated with Her-2 status, TNM stage and lymph node metastasis, and the target genes of miR-21-3p were involved in the proteasome-mediated ubiquitin-dependent protein catabolism, Ras signaling pathway and chemokine signaling pathway and other key pathways and processes related to cancer progression. Conclusion Serum miR-21-3p level detection has certain clinical guiding significance for the diagnosis of breast cancer, and is expected to become a new biomarker for the diagnosis of breast cancer