The invention relates to a method used for in vitro proliferation of NK cells, and more specifically relates to a method used for realizing large-scaled proliferation of NK cells via autologous peripheral blood mononuclear cell in vitro cultivating. According to the method, proliferation and activation of NK cells are realized via different cytokine combination. The method comprises following steps: autologous peripheral blood mononuclear cells are inoculated in CD3 antibody and CD16 antibody pre-coated culture bottles, and are subjected to stimulation for 16 to 24h; and a serum-free medium containing IL-2 and IL-18 is added. According to the method, CD3 antibody and CD16 antibody are immobilized via coating, direct stimulating activation of NK cells is performed, so that toxicity of NK cells is increased greatly, activation and proliferation of NK cells are realized using two cytokines IL-2 and IL-18, proliferation multiple and cytotoxicity of NK cells are ensured, and culturing cost of NK cells is reduced greatly.