The invention discloses a DC-CIK cell culture medium, a DC-CIK cell culture method and application, and relates to the technical field of cell culture. The DC-CIK cell culture medium comprises a DC culture medium containing specific components, a DC maturation factor, a CIK promoter, a CIK bottle enlargement culture medium, a CIK separate culture medium, a coating liquid and a basic culture medium, and all the components have different effects through different compositions. When the DC-CIK cell culture medium is used for culturing the DC-CIK cells, the number of the obtained DC-CIK cells is substantially increased and can reach (1.05-1.56) * 1011/mL, the survival rate is kept at 96.6%-99.7%, meanwhile, the CD3+, CD4+ and CD5+ of the DC-CIK cells can reach 68%.