Abstract: Objective To explore the value of serum semaphorin 4F (SEMA4F) combined with carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), and carbohydrate antigen 19-9 (CA19-9) in the diagnosis of breast cancer. Methods A total of 111 breast cancer patients who underwent surgical treatment at Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University from August 2023 to October 2024 were selected as a breast cancer group. Moreover, 70 patients with benign breast diseases were selected as a benign breast disease group, and 50 healthy women who underwent physical examinations were selected as a healthy control group. The serum levels of SEMA4F, CEA, CA125, CA15-3, and CA19-9 were measured and compared in each group using ELISA. The relationship between serum SEMA4F and the clinical pathological characteristics in breast cancer patients was analyzed. Receiver operating characteristic (ROC) curves were plotted to evaluate the diagnostic efficacy of SEMA4F combined with traditional breast cancer tumor markers in the diagnosis of breast cancer. Results Compared with the benign breast disease group and the healthy control group, the levels of serum SEMA4F, CEA, CA125, CA15-3, and CA19-9 were significantly elevated in the breast cancer group (P<0.05). The serum SEMA4F level in breast cancer patients was associated with pathological type, tumor size, lymph node metastasis, distant metastasis, and TNM staging (P<0.05), rather than estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), Ki67, or molecular subtypes (P>0.05). ROC curve analysis showed that the sensitivity and specificity of diagnosis of breast cancer using SEMA4F combined with CEA, CA125, CA15-3, and CA19-9 were 86.50% and 94.20%, respectively. Conclusions Serum SEMA4F combined with CEA, CA125, CA15-3, and CA19-9 is of importance in the early diagnosis of breast cancer.