Objective To investigate the clinical value of serum anti-centromere protein F antibody（anti-CENPF） in breast cancer. Methods Serum samples were collected from 100 breast cancer patients（BC group），40 patients with benign breast disease（non-BC group），and 40 healthy controls（HC group）. Enzyme-linked immunosorbent assay was used to measure the serum level of anti-CENPF，and chemiluminescence was used to measure the serum level of carbohydrate antigen 153（CA153）. Results The BC group had a significantly higher serum level of anti-CENPF than the non-BC group and the HC group（P<0.05）. The receiver operating characteristic（ROC） curve analysis showed that anti-CENPF and CA153 had an area under the ROC curve（AUC） of 0.714 and 0.672，respectively，in the diagnosis of breast cancer，and the combination of anti-CENPF and CA153 had an AUC of 0.739. There was a significant increase in the concentration of serum anti-CENPF in breast cancer patients with lymph node metastasis，distant metastasis，or negative human epidermal growth factor receptor 2（HER-2）（P<0.05）. There was a significant difference in the serum level of anti-CENPF between breast cancer patients with different tumor sizes，clinical stages，and molecular types（P<0.05）. Comparison between groups showed that patients with stage Ⅲ/Ⅳ breast cancer had a significantly higher serum level of anti-CENPF than those with stage Ⅰ/Ⅱ breast cancer，and patients with HER-2 overexpression or Luminal B type breast cancer had a significantly lower serum level of anti-CENPF than those with triple negative breast cancer. Among patients with estrogen receptor-positive breast cancer，the HER-2 negative group had a significantly higher concentration of anti-CENPF than the HER-2 positive group（P=0.026）. Conclusion Serum anti-CENPF plays an important role in the diagnosis，clinical staging，and molecular typing of breast cancer，and the level of anti-CENPF may be negatively correlated with the prognosis of breast cancer. Therefore，it is expected to become a potential disease marker for breast cancer.