Abstract: The present disclosure provides an in vitro proliferation medium, an in vitro culture kit and an in vitro proliferation culture method of umbilical cord blood (UCB)-derived natural killer (NK) cells, and relates to the technical field of cell in vitro culture. In the present disclosure, the in vitro proliferation medium does not include animal serum to reduce immune responses when being used, with better safety. The in vitro proliferation medium is used to proliferate the UCB-derived NK cells in vitro, with simple and easy operations and low cost, and without coating, sorting and trophoblast cells. The medium does not include animal-derived ingredients, and has desirable safety and stability. The medium can be used for direct culture of peripheral blood mononuclear cells (PBMCs) isolated by Ficoll, and harvest more NK cells that meet clinical use standards.