Since African swine fever (ASF) was first reported in 1921, it has brought huge economic losses to the world pig industry. No vaccine or therapy is available. Rapid and effective diagnostics are key steps in managing ASF. We generated three monoclonal antibodies (mAbs) against the African swine fever virus (ASFV) phosphoprotein p30 and designated these as 7D2, 8C8 and 2F6. Epitope mapping revealed that mAb 7D2 recognized ²⁶VFHAG SLYNW ³⁵ of p30, and mAb 8C8 and 2F6 recognized ¹MDFIL NISMK MEVIF KTDLR ²⁰ of p30. Furthermore, epitope ¹MDFIL NISMK MEVIF KTDLR ²⁰ and ²⁶VFHAG SLYNW ³⁵ could be well recognized by ASFV-positive sera from natural infected pigs, suggesting that they were natural linear B-cell epitope. Conservation analysis indicated that epitope ¹MDFIL NISMK MEVIF KTDLR ²⁰ and ²⁶VFHAG SLYNW ³⁵ were highly conserved among the different strains of ASFV. This is the first research to characterize specific mAbs against p30 protein. These findings may facilitate further understanding the function of p30 protein and development of diagnostic tools.