Abstract: In order to obtain hybridoma cells secreting monoclonal antibody (McAb) direct against Reticuloendotheliosis Virus (REV), BALB/c mice was immunized with the purified gp90 as immunogen, then hybridoma cell strains were screened for the secretion of McAb against REV. Two hybridoma strains with the ability to stably secrete McAb against REV, designated as A9E8 and B3F8 respectively, were obtained after threeround screening. The titers of secreting antibody from cell culture supernatant were both above 1∶4×102, while above 1∶8×103 from ascites of the mice. The secreted McAbs were both IgG, and the light chains were both κ as indicated by antibody subtype identification assay. IFA results showed that secreted McAb could react specifically with the REV strain. Applying the property, an indirect immunofluorescent method for the REV antigen was established, and the actual application result showed the diagnosis was nearly in accordance with the result of virus isolation and PCR, and the positive according rate was 90%, the negative according rate was 100%. Hybridomas trains that secrete McAb specific against REV were produced, which will be useful tools for REV rapid diagnosis and immunogenic analysis of the protein such as epitope mapping.