Heat shock protein 90α (HSP90α) has been confirmed to be upregulated in the blood in various types of tumors and may therefore serve as a potential tumor marker. However, whether HSP90α exists in nipple discharge remains unknown, and its expression and diagnostic value in nipple discharge remain unclear. In this study, the expression of HSP90α, carcinoembryonic antigen (CEA), and cancer antigen 153 in nipple discharge and blood from 128 patients was measured. Receiver operating characteristic curve was used to assess the diagnostic value of HSP90α. Further, its relationship with clinicopathological parameters of patients with breast cancer was analyzed. The results showed that the expression of HSP90α in nipple discharge was significantly higher in patients with breast cancer than in those with benign disease, and its diagnostic value was better than that of CEA. Combination of HSP90α and CEA showed better diagnostic efficacy than HSP90α or CEA alone. Moreover, the expression of HSP90α displayed a stepwise increase from benign lesions, followed by carcinoma in situ to invasive ductal carcinoma. HSP90α was positively correlated with Ki67 expression. However, there was no significant difference in the expression of HSP90α in blood between patients with breast cancer and benign disease. Further, the expression of HSP90α was higher in nipple discharge than in blood. In summary, HSP90α was upregulated in the nipple discharge of patients with breast cancer, and it may be related to the occurrence and progression of breast cancer. HSP90α in nipple discharge may serve as a potential diagnostic marker for breast cancer.