The invention discloses a cell line for stable expression of a chicken interleukin 10 protein, as well as a construction method and application thereof. The construction method disclosed by the invention comprises the steps of cloning a chicken interleukin 10 encoding gene after removal of a signal peptide into a eukaryotic expression vector containing a neomycin resistance gene in a fixed direction to obtain a recombinant plasmid, transfecting cells of a Chinese hamster ovary cell line CHO-K1, and performing G418 pressurized screening and purification to obtain a cell line CHO-chIL-10M for stable and high-efficient expression of a chIL-10 protein, wherein the microorganism collection number is CGMCC NO. 7659. The cell line CHO-chIL-10M constructed by the construction method disclosed by the invention has extensive application prospects in preparation of recombinant chicken interleukin 10 proteins and chicken interleukin 10 monoclonal antibodies and other aspects.