Objective: MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that can regulate gene expression, thereby affecting crucial processes in cancer development. miRNAs offer great potential as biomarkers for cancer detection because of their remarkable stability in blood and their characteristic expression in different diseases. In the current study, we focused on Mir-382, one potential tumor-associated miRNA, investigated whether Mir-382 on serum could discriminate between breast cancer patients and healthy controls. Methods: We performed miRNA quantitation assay on serum from breast cancer patients, followed by construction of ROC (Receiver Operating Characteristic) curves to determine the sensitivity and specificity of the assay. A total of 164 breast cancer patients who underwent tumor resection were included in our follow-up study, meanwhile same number healthy subjects were also enrolled as control. Results: In this study, we aimed to investigate whether Mir-382 is associated with the TNM staging and prognosis of breast patients. We found that the Mir-382 expression was significantly lower in tumor tissues compared with that in normal tissue (P<0.05). Kaplan-Meier and analysis showed that the Mir-382 expression status is significantly associated with the survival duration (Log-rank test, P<0.001), and multivariate Cox regression revealed that patients with low Mir-382 expression were exposed to a 2.352 fold higher death risk (HR=2.352, 95% CI=1.379-4.012, P=0.002) compared with those with high Mir-382 expression. Expression of MiR-382 was also significantly correlated with TNM staging, especially for lymph node metastasis and distant metastasis. By in vitro study, exogenous expression Mir-382 could significantly inhibit the proliferation and migration of breast cancer cell MCF-7; on the other hand, knocking down Mir-382 could significantly reverse these biological characteristics. Conclusion: Our study thus demonstrates that Mir-382 expression in breast tissues is critically associated with the prognosis of patients, suggesting its potential clinical significance.