The invention discloses a method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity. The method comprises the following steps: a, taking fresh human
peripheral blood and obtaining mononuclear cells with a
gradient centrifugation method; b, pre-stimulating mononuclear cells in an
in vitro medium; c, expanding culture of
peripheral blood derived mononuclear cells, detecting the proportion of various cells, and judging amplification efficiency and activity of the target NK / NKT cells; d, detecting the proportion of NK / NKT cells to the
cell population after culture for 3-4 weeks, and detecting
cell killing efficiency; e, further purifying the NK / NKT cells. The method is simple to operate, the NK
cell population rich in NKT cells with high killing activity is obtained and contains no
impurity cells,
immunogenicity caused by the
impurity cells is avoided, and side effects in clinical application are lower. Besides, the method has the advantages that materials are easily available, the cells are suitable for large-scale culture, damage to the body is small and the like, and is more suitable for
adjuvant treatment of tumor patients.