Interleukin-8(IL-8)/CXCL8 is a CXC-family chemokine that attracts lymphocytes to sites of tissue damage and plays a role in the inflammatory response and wound healing. Chicken chemotactic and angiogenic factor was referred to as chCXCLi2 and has been studied as one of human CXCL8 homologue for more than 20 years. However, no monoclonal antibodies (mAbs) that specifically detect chCXCLi2 have been developed. Here, we developed and characterized mouse mAbs against chCXCLi2 to define its immunological properties. Two mouse mAbs against chCXCLi2 were generated and confirmed to display specific binding with not only recombinants, but endogenous chCXCLi2 by Western blot analysis, ELISA, and immunocytochemistry. Inhibition of chCXCLi2-induced chemotactic activity on peripheral blood lymphocytes, proliferation of chicken macrophage cells and expression of alpha smooth-muscle actin in chicken embryonic fibroblast cells by antibodies indicate that these antibodies are capable of blocking chCXCLi2 bioactivity. These chCXCLi2 mAbs will be useful reagents for future investigations of inflammation in poultry.