Abstract: In order to prepare a monoclonal antibody against the pB602 L protein of African swine fever virus（ASFV）,the E.coli expression system was used to express the recombinant pB602 L protein for the immunization on Balb/c mice. Through cell fusion and subcloning, hybridoma cell lines stably secreting monoclonal antibodies against ASFV pB602 L protein were obtained, and monoclonal antibodies were prepared by intraperitoneal injection of cells into mice. The titer was determined by indirect ELISA method, the monoclonal antibody subtype identification kit was used to determine its subtype, Western-blot was used to identify its specificity, and the ASFV antigen-coated plate was used to identify its reactivity. The results showed that 17 hybridoma cell lines that stably secreted monoclonal antibodies against ASFV pB602 L protein were obtained, and the ascites titer was not less than 1∶1 280 000. The heavy chain subclasses were IgG1,IgG2 a and IgG2 b, and the light chain was κ. Those antibodies can specifically recognize recombinant pB602 L protein, and there were 16 monoclonal antibodies which had different degrees of antigen-antibody reaction with ASFV antigen, and the OD₄₅₀ value of the reaction can reach up to 1.098. The results suggested that the ASFV pB602 L protein monoclonal antibodies prepared for the first time had good specificity and can react with viral antigen.