The invention relates to NK cells with enhanced killing activity and a preparation method thereof. The method includes the steps that a, feeder cells are prepared from 
peripheral blood mononuclear cells PBMCs; b, 
NK cell proliferation is conducted, wherein in the step a, the PBMCs are primarily stimulated with 
monoclonal antibodies CD3 and continue to be activated in a first culture medium containing IL-2, IFN-r, IL-15 and IL-18, primarily activated PBMCs with 
antigen presentation are obtained and irradiated with gamma rays to be used as the feeder cells, and part of the feeder cells are subjected to freeze preservation with 
liquid nitrogen; in the step b, CD3+T lymphocytes are removed from the original PBMCs not activated through an 
immunomagnetic bead sorting method, NK cells after CD56+are gathered again selectively, the NK cells and the feeder cells not subjected to freeze preservation with 
liquid nitrogen are subjected mixed 
hatching, 
NK cell proliferation culturing is conductedin a second culture medium containing IL-2, IL-15, OK432 and 
nicotinamide, then the NK cells are transferred into a third culture medium containing IL-2 and 
nicotinamide to be subjected to proliferation culturing, and finally the feeder cells which are thawed from 
liquid nitrogen freeze preservation are added to continue proliferation culturing.