The invention relates to an NK cell in-vitro amplification culture method, and belongs to the field of cell culture. The NK cell in-vitro amplification culture method comprises the following steps: screening out a large number of T cells by using a CD 16-coated culture bottle, so that NK cells become dominant cell populations; stimulating and inducing the NK cells by using IL-2, IL-12, IL-15, IL-18, SCM and IFN-gamma; and amplifying by using a serum-free complete medium containing IL-2 to obtain a large number of high-purity NK cells. The problem that a large number of high-purity NK cells cannot be obtained by amplification in the prior art is solved. According to the method, most of T cells are screened out by using the CD 16-coated culture bottle, so that the NK cells become dominant cells, and the NK cells are stimulated, induced and amplified; the method is simple to operate, easy to implement and high in amplification efficiency, no feeder cells or conductor serum is added in theculture process, the medical ethics and GVHD risk problems cannot be caused, and the method is suitable for clinical experimental research and large-scale preparation of the NK cells.