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Avian leukosis virus subgroup J ( ALV-J ) is an avian oncogenic retrovirus that has caused huge economic losses in the poultry industry due to its great pathogenicity and transmission ability. However, the continuous emergence of new strains would bring challenges to diagnosis and control of ALV-J. .This study focuses on preparing the monoclonal antibody ( MAb ) against ALV-J Gp85 and identifying its epitope. The truncated ALV-J gp85 gene fragment was amplified and then cloned into expression vectors. Purified GST-Gp85 was used to immune mice and His-Gp85 was used to screen MAb. Finally, a hybridoma cell line named J16 that produced specific MAb against the ALV-J. Immunofluorescence assay showed that MAb J16 specifically recognized ALV-J rather than ALV-A or ALV-K infected DF-1 cells. To identify the epitope recognized by MAb J16, fourteen partially overlapping ALV-J Gp85 fragments were prepared and tested by Western blot. The results indicated that peptide 150-LIRPYVNQ-157 was the minimal epitope of ALV-J Gp85 recognized by MAb J16. Alignment analysis of Gp85 from different ALV subgroups showed that the epitope keep high conservation among 36 ALV-J strains, but significant different from that of ALV subgroup A, B, C, D, E and K. Overall, we prepared a MAb specific against ALV-J and identified peptide 150-LIRPYVNQ-157 as a novel specific epitope of ALV-J Gp85, which may assist in laying the foundation for specific ALV-J detection methods.