Type Ⅱ interferon, also termed IFN-γ (IFN-γ), is one of the key cytokines involved in the immune response of T helper 1 (Th1) lymphocytes. Fish and mammalian type Ⅱ IFN-γs are homologs, but unlike mammals which contain a single copy of IFN-γ gene, teleost fish have two duplicated copies, namely IFN-γ and IFN-γ related (IFN-γrel) factor. To date, the expression and functions of fish IFN-γs have been well documented, however fish IFN-γrels have not been extensively investigated. For example, the cells producing IFN-γ and IFN-γrel are unclear, whether they have differences in functions has also been debated. In this study, the IFN-γ and IFN-γrel recombinant proteins of Ctenopharyngodon idella (Ci) were expressed in E. coli DE3 cells. The results showed that the CiIFN-γ protein was soluble, while the CiIFN-γrel protein was insoluble. The recombinant IFN-γ and IFN-γrel proteins were purified using affinity chromatography and size exclusion chromatography and used for preparation of monoclonal antibodies in mice. Four antibodies with high specificity and affinity were obtained after screening by Western blotting. It was revealed that the monoclonal CiIFN-γ antibodies did not cross-react with the CiIFN-γrel protein and vice versa. Two of the antibodies were labeled with FITC fluorescein and could be used for immunofluorescent analysis and flow cytometry. Further, C. idella IFN-γ and IFN-γrel monoclonal antibodies could not recognize their respective homologs from Danio rerio. To our knowledge, this is the first study reporting the preparation of IFN-γrel monoclonal antibody in fish. The availability of CiIFN-γ and CiIFN-γrel monoclonal antibodies will provide valuable information to investigate the cellular sources and biological functions of type Ⅱ IFNs in C. idella.