The experiment was to obtain the ASFV structural protein VP73 that was expressed in vitro, which was as a diagnostic antigen. According to the gene sequence of ASFV VP73 in the gene bank, the major antigenic epitope gene of VP73 was synthesized and cloned into pUC57 vector. The VP73 gene of African Swine Fever Virus was amplified from DNA by PCR, the product of which was a 429 bp DNA segment. The purified VP73 gene was subcloned into pGEXKG vector by EcoRI and Sal enzymes digestion. The recombinant plasmid was identified by PCR and enzymes digestion. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted VP73 gene. The recombinant plasmid was transformed into BL21 (DM3).The recombinant protein was induced expression by IPTG which was analyzed by SDSPAEG and Western blot. The results show the recombinant plasmid was successfully built. The results of SDSPAGE revealed that the VP73 protein was expressed in pGEXKG vector. The optimal amount of the expressed fusion protein was 35% of total bacterial protein. It effectively expressed 41Ku recombinant protein and the Western blot analysis indicated a better immunologically reactive activity. The experiment has laid a foundation for the establishment of a rapid, sensitive serologically diagnostic method which is free of infectivity.