Objective To study the diagnostic value of serum Thymosin β4 level on chemotherapy-related cardio-toxicity of breast cancer. Methods The double antibody sandwich ELISA was used to detect serum Tβ4 level in 129 breast cancer patients and analysis if ECG was associated with chemotherapy-related cardio-toxicity. The sensitivity and specificity of ECG in the diagnosis the cardio-toxicity was assessed. Results A total of 18 patients (13.95%) had subsequent claims for cardio-toxicity events. Tβ4 in cardio-toxicity group was significantly higher than that non-cardio-toxicity group (P <0.01). According to ROC curve, the cut off value of Tβ4 was set at 6000pg/mL, the specificity and sensitivity were 72.22％ and 85.60％ respectively．Conclusion The present study is to confirm excellent clinical value of Tβ4 on cardio-toxicity. The level of Tβ4 for early detection of cardio-toxicity has good prospects for high-risk patients.