Cell culture medium is an important factor affecting the expansion of NK cells in vitro. As an important component of cell culture medium, lipids participate in various complex physiological activities of cells and significantly affect the expansion of cells. Using NK-92 cells as a model, the lipid metabolism of NK cells in vitro was analyzed, and combined with the kinetic relationship between lipid metabolism and NK cell expansion. Four fatty acids, oleic acid, linoleic acid, palmitoleic acid, and α-linolenic acid, were preliminatively identified as the key lipid combinations. The combination was preliminarily verified on the self-developed serum-free medium. It was found that when the key lipid combination was added according to the concentration in the serum, NK-92 cells expansion reached 188.03 ± 33.34-folds, which was significantly higher than 105.28 ± 13.23-folds in the basic medium. Additionally, NK-92 cells expanded by adding key lipid combinations could maintain cell killing function. Overall, this research provides technical support for the development of NK cell serum-free medium.