African swine fever virus (ASFV) is a complex DNA virus belonging to the family Asfarviridae. The outbreak of African swine fever (ASF) has caused huge economic losses to the pig farming industry. The K205R protein is a key target for detecting ASFV antibodies and represents an important antigen for early serologic diagnosis. In this study, we obtained soluble K205R protein in the E. coli expression system. Furthermore, we prepared monoclonal antibodies (mAbs) 6F5 and 6E2 using cell fusion technique, and verified their specific recognition ability for recombinant ASFV K205R protein expressed in prokaryotic and eukaryotic cells using protein immunoblotting and indirect immunofluorescence. Using the truncated overlapping peptide method, 6F5 and 6E2 specifically recognized