The diagnostic efficacy of carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA15-3) is limited in breast cancer (BC), highlighting the necessity of exploring novel biomarkers to improve for BC diagnosis. Therefore, we assessed the diagnostic value of fat mass and obesity-associated protein (FTO), phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit β (PIK3CB) as a potential complementary biomarker to CEA and CA153 in breast cancer by measuring serum FTO,PIK3CB levels. FTO, PIK3CB, CEA and CA15-3 levels were measured in 112 BC patients and 64 healthy controls using enzyme-linked immunosorbent assay or electrochemiluminescence immunoassay. Spearman’s rank correlation analysis was conducted to assess the correlation between the levels of the 2 markers. The relationships between FTO, PIK3CB, CEA, CA15-3 and clinical characteristics were evaluated. Receiver operating characteristic curve (ROC) analysis was performed to assess the diagnostic value of FTO, PIK3CB, CEA and CA15-3 of BC. Serum FTO, PIK3CB, CEA and CA15-3 levels were significantly increased in BC. There was no correlation between FTO, PIK3CB and CEA, CA15-3. FTO and PIK3CB demonstrated significant diagnostic performance for breast cancer, with FTO achieving a specificity of 90.63%. The diagnostic performance of 2-four biomarker combinations was significantly superior to individual CEA or CA153, with a combined panel of 4 biomarkers yielding an area under the curve (AUC) of 0.918, sensitivity of 81.25% and specificity of 85.94%. In early-stage breast cancer (I + II), the combination of FTO, PIK3CB, CEA and CA153 yielded an AUC of 0.895, sensitivity of 77.22% and specificity of 85.71%. FTO and PIK3CB can be served as potential biomarkers to complement CEA and CA15-3 for BC diagnosis. Combining FTO, PIK3CB, CEA and CA15-3 improves the diagnostic efficiency of breast cancer.