The invention provides a natural killer T cell culture medium and a multiplication culture method for natural killer T cells and relates to the technical field of cell culture. The natural killer T cell culture medium, disclosed by the invention, is mainly composed of a serum-free medium, autologous plasma and a cell factor IL-2. According to the multiplication culture method for natural killer T cells by using the cell culture medium, human-derived peripheral blood mononuclear cells are induced to release a danger signal by virtue of the antibody binding cell factor IL-2, and further the natural killer T cells are activated. According to the method, high-quality natural killer T cells can be obtained by massive high-efficiency multiplication in a short period on premise of ensuring the security. The potential security risk caused by culture of heterogenous animal serum or tumor cells in the conventional natural killer T cell culture method and the problems that the conventional natural killer T cell culture method is low in multiplication times and low in purity and is difficult to use on a large scale are effectively solved.