African swine fever virus (ASFV), a highly contagious virus with a double-stranded DNA genome, is notorious for causing severe hemorrhagic fever in pigs, often leading to mortality rates as high as 100 %. First identified in Kenya in 1921, the virus has since spread globally, with a significant outbreak in China in 2018, causing extensive economic losses in the swine industry. The D205R protein (pD205R) of ASFV, classified as a non-structural protein, plays a role in the transcription of viral genes and is associated with ASFV RNA polymerase. However, the specific function of this protein remains unclear. To gain a deeper insight into the structure, function, and mechanisms of interaction between pD205R and the host, we successfully expressed the pD205R protein and generated a monoclonal antibody (mAb), designated 3G6G1, targeting this protein. The mAb 3G6G1 can be utilized for indirect immunofluorescence (IFA) and Western blotting (WB) detection of ASFV strains. Through the evaluation of the reactivity of antibodies against a series of truncated pD205R fragments, we identified the epitope recognized by mAb 3G6G1 as residing within the amino acid sequence 96 VLSKKNI 102. Bioinformatics analysis indicated that this antigenic epitope possesses a high antigenic index and is highly conserved. These findings will establish a foundation for further research into the function of the D205R protein and its role in the interaction between ASFV and its host.