Background: African swine fever (ASF), characterized by acute, severe, and fast-spreading, is a highly lethal swine infectious disease caused by the African swine fever virus (ASFV), which has caused substantial economic losses to the pig industry worldwide in the past 100 years. Methods: This study started with bioinformatics methods and verifed the epitope fusion protein method’s reliabil‑ ity that does not rely on traditional epitope identifcation. Meanwhile, it will also express and purify the constructed genes through prokaryotic expression and establish antibody detection methods. Results: The results indicated that the protein had good reactivity and did not cross-react with other swine diseases. The receiver-operating characteristic analysis was performed to verify the determination. The area under the receiveroperating characteristic curve was 0.9991 (95% confdence interval 0.9973 to 1.001). Conclusions: It was proved that the recombinant protein is feasible as a diagnostic antigen to distinguish ASFV and provides a new idea for ASFV antibody detection.