African swine fever (ASF) is one of the most important infectious diseases of wild and domestic pigs. To develop safe and efficient diagnostic tools for ASF, we amplified the CP204L and E183L genes, which encode the major antigenic proteins p32 and p54, respectively, of the ASF virus Lisbon′60BM89BC1 strain by polymerase chain reaction and expressed them in recombinant baculoviruses. Each recombinant protein was recognized as a single band in baculovirus-infected insect cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific reaction was observed between these recombinant proteins and test sera from pigs infected with several strains of ASF virus in Western blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). In Western blot analyses, p54 showed relatively higher sensitivity than p32 in antibody detection in the early stage of infection. On the other hand, p32 exhibited better reactivity in indirect ELISA to discriminate between positive and negative reactions. These data indicate that the recombinant p32 and p54 are useful as diagnostic reagents for serological tests of ASF. In order to diagnose ASF accurately, both recombinant proteins should be used in Western blot analysis and indirect ELISA.