Breast cancer is the second leading cause of cancer death in women. Serum biomarkers such as cancer antigen 15-3 (CA15-3), cancer antigen 125 (CA125), and carcinoembryonic antigen (CEA) can be used as diagnostic and prognostic factors and can also provide valuable information during follow-up. However, serum protein biomarkers show limited diagnostic sensitivity and specificity in stand-alone assays because their levels reflect tumor burden. To validate whether biomarkers in nipple discharge may serve as novel biomarkers for breast cancer, we composed a panel of potential cancer biomarkers, including CA15-3, CA125, CEA, and malignant tumor-specific growth factor (TSGF), and evaluated their expression in both serum and nipple discharge in order to explore the expression and significance of estrogen receptor (ER), progestrone receptor (PR), epidermal growth factor receptor type 2 (HER2/neu), CA15-3, CA125, CEA, and TSGF expression for their combined predictive value for breast cancer and in judging the prognosis of breast cancer. Univariate analysis revealed that combined detection of CA15-3, CA125, CEA, and TSGF in nipple discharge served as novel biomarkers for the diagnosis and prognosis of breast cancer, but in the multivariate analyses the adverse effects of the four biomarkers combination in nipple discharge positivity on overall survival were lost. Multivariate analysis revealed that the positivity of the combined detection of the four biomarkers in both nipple discharge and serum was significantly higher than that of other detection methods. Thus, the combined detection of these four biomarkers both in serum and nipple discharge was retained as an independent prognostic variable in breast cancer patients. Our results indicate that CA15-3, CA125, CEA, and TSGF in nipple discharge can serve as novel biomarkers in the diagnosis and prognosis of breast cancer.