The cyprinid herpesvirus Ⅱ (CyHV-2) ORF25 gene mainly encode membrane proteins,which show good antigenicity by antigen eptitope prediction.In this study,ORF25 gene segment was amplified by PCR and cloned into the prokaryotic expression vector pGEX-KG.The CyHV-2 ORF25-KG fusion protein (42 ku) was expressed highly under induction of IPTG in the E. coli BL21,and this fusion protein was mainly expressed in the insoluble composition.One monoclonal antibody (McAb 5C6) against ORF25 protein were generated by fusion of mouse myeloma cell line SP2/0 and spleen lymphocytes from immunized mice.The McAb 5C6 was further characterized by IFA (indirect immunofluorescent assay) and Western blot assay.This study laid a foundation for the research of the pathogenesis and the detection method of CyHV-2.