The invention discloses an in-vitro culture method of NK (natural killer) cells and belongs to culture of human cells. The in-vitro culture method disclosed by the invention comprises the following steps: merging herceptin diluted by PBS (phosphate buffered saline) and human immunoglobulin diluted by the PBS, then uniformly and fully spreading at the bottom of a culture bottle and standing overnight; additionally taking peripheral blood, performing density gradient centrifugation, sucking a single nuclear cell, adding into a serum-free culture medium, and adjusting the concentration of the cells to 1.0*10<6> / ml-3.0*10<6> / ml; and then adding cell factors IL-2 and IL-15, adding into the culture bottle coated by the herceptin and culturing in an incubator. Therefore, on the basis of ensuring the amplification multiple of various cell subgroups, the growth and the proliferation of the NK cells are promoted, the killing activity of lymphocytes is enhanced, the serum-free culture medium can replace a serum-containing complete culture medium, the number of obtained culture products is equivalent to the activity of the cells, the in-vitro large-scale culture of the NK cells is realized, the in-vitro culture method is used for clinical biological treatment of the NK cells, and the safety in clinical application can be increased by using the in-vitro culture method.