Abstract: Interleukin (IL) 10 is synthesised and secreted mainly by type 2 innate lymphoid cells (ILC2) and T helper 2 cells (Th2) and regulates the growth, proliferation and differentiation of immune cells such as T cells, B cells, monocytes/macrophages and dendritic cells. It plays an important role in regulating immune response and maintaining immune homeostasis by synergizing with other cytokines. In this study, a plasmid pET-21d-CiIL-10 was constructed for expression of the recombinant protein in the Escherichia coli Rosetta (DE3) cells. After induction with IPTG, the recombinant protein of grass carp (Ctenopharyngodon idella, Ci) IL-10 was found in the inclusion bodies. After denaturation with 6M guanidine hydrochloride and refolding, the CiIL-10 protein was purified by size exclusion chromatography and analyzed by SDS-PAGE. The results showed that a single band of the CiIL-10 protein was visualized, confirming that the protein was pure. The recombinant CiIL-10 protein was then used to immunize mice to generate monoclonal antibodies. Four monoclonal antibodies were obtained, among which two could recognize the CiIL-10 protein expressed in the HEK293 cells. One monoclonal antibody was selected for purification and further analyzed by confocal microscopy and flow cytometry. It was shown that the CiIL-10 monoclonal antibody could react with the recombinant CiIL-10 protein expressed in the E. coli cells and HEK293 cells. In addition, confocal microscopic analysis revealed that the monoclonal antibody could detect the native CiIL-10 protein in the grass carp ovary (GCO) cells treated with PBS, lipopolysaccharide (LPS) or CiIL-1β for 36h. Interestingly, compared to the control group (PBS), the numbers of CiIL-10 producing cells remained largely unchanged after stimulation with LPS or CiIL-1β. In summary, high purity of the recombinant CiIL-10 protein was obtained from the E. coli cells and the CiIL-10 monoclonal antibody generated is highly specific. It is expected that the CiIL-10 monoclonal antibody will have great potential to be used in detection of IL-10 in grass carp by Western blotting, enzyme linked immunosorbent assay (ELISA), flow cytometry and confocal microscopy.