Background. Chemotherapy-induced amenorrhea (CIA) is one of universal phenomena in breast cancer (BC) patients, and it causes difficulties in evaluating the actual menopausal status which is important for the oncologists to choose appropriate treatment. Currently, serum estradiol (E2) and follicle-stimulating hormone (FSH) levels are the most commonly used clinical parameters for the assessment of menopausal status in BC patients. However, the optimal cut-off points of serum E2 and FSH have little been explored in southern Chinese population. Objective. This study is aimed to determine the optimal cut-off values of the serum E2 and FSH levels for evaluating the menopausal status of BC patients in a southern Chinese population. Methods. A retrospective analysis was done among a total of 206 patients with BC from a southern Chinese area. The data of serum E2, FSH, and luteinizing hormone (LH) levels were collected and analyzed for the comparison purpose. The receiver-operating curve (ROC) was generated to assess the specificity and sensitivity of the three biomarkers in discriminating the menopausal status of BC patients. The optimal cut-off values were determined according to the Youden index and then compared with the recommended reference values by the Chinese Anti-cancer Association (CACA) and those recommended by the manufacturers. Results. The areas under the ROC curves (AUCs) of E2, FSH, and LH were 0.846 (95% CI: 0.790-0.903), 0.781 (95% CI: 0.714-0.847) and 0.608 (95% CI: 0.526-0.690), respectively. The optimal cut-off values were 130.0 pg/mL for E2, 23.325 IU/L for FSH, and 11.625 IU/L for LH with a maximum of the Youden index. When E2, FSH, and LH were used in combination for ROC analysis, the AUC increased to 0.847 (95% CI: 0.790-0.904), which was higher than that of any other biomarker alone. In this study, the sensitivity and specificity of E2 and FSH were 91.6% and 73.70% and 94.4% and 58.6%, respectively, in comparison with 85.0% and 75.80% and 76.6% and 65.7% according to the CACA-recommended cut-off points, or 92.5% and 68.7% and 96.3% and 53.5% according to the manufacturer recommended cut-off points. Conclusion. Considering the sensitivity and specificity of serum E2 and FSH for assessing the menopausal status, the optimal cut-off values determined in the present study were similar to the manufacturer’s recommendations, but obviously superior to the cut-off points suggested by CACA. These cut-off points calculated in this study seem to be valuable in southern Chinese population and might be used by clinicians to make a correct medical decision for BC patients who would benefit from endocrine therapy of aromatase inhibitor (AI).