Objective To investigate the expression of miR-181a and ATG5 in breast cancer patients, and further study their regulatory effects on autophagy of MCF-7 breast cancer cell line, in order to reveal their clinical significance and potential mechanism in the development of breast cancer. Methods In this study, 120 patients with breast cancer admitted from June 2021 to September 2022 were selected as disease group and 55 healthy subjects were selected as normal control group. The expression of miR-181a and ATG5 in serum and tissues was detected by qPCR. CCK-8 was used to detect the changes in the proliferation ability of MCF-7 cells, and Western blot was used to detect the expression of autophagy and apoptosis-related proteins in MCF-7 cells. The apoptosis rate of MCF-7 cells in each group was detected by flow cytometry. Results The expression of miR-181a in serum and tissues of breast cancer patients was significantly increased, while the expression of ATG5 was significantly decreased (P<0.01). The expression of miR-181a was closely related to lymph node metastasis, TNM stage and tumor differentiation (P<0.01). Inhibition of miR-181a expression could significantly reduce the proliferation capacity of MCF-7 cells, promote the expression of autophagy related proteins ATG5, LC3B and Beclin-1, and promote the expression of apoptotic BAX/ BCL-2 protein ratio. Conclusion The up-regulation of miR-181a is associated with the clinical characteristics of breast cancer and may be used as a potential marker for disease assessment. In addition, miR-181a may affect the proliferation and survival of breast cancer cells by regulating autophagy and apoptosis. It is expected to provide a new research direction and clinical application prospect for the diagnosis and treatment of breast cancer