The invention discloses a method for preparing an antitumor adoptive immunocompetent cell CIK (cytokine induced killer). The problems of CIK cells prepared in the prior art that the multiplication capacity of the cells is low, the quantity of obtained effector cell mass is limited, and the cell culture cycle is long are solved. According to CIK cells prepared by the method, CIK preliminary cells are treated through combined application of L-6 and IL-21, thus, the quantity of Treg cells is reduced, and the quantity and tumor killing capability of effector cells are relatively increased; subsequently, CD3mAb is replaced with TG, so that the amplification, differentiation and aging of CD3+CD56+cell mass are improved in an aimed manner. Furthermore, TG of a clinical application level is an optimization and completion of a CIK preparation system in safety and effectiveness at present. Finally, through culturing the CIK cells in an IL-27 and IL-2 combined manner, the quantity and efficacy of the finally prepared CIK cells are further improved, adverse reactions of patients caused by cytokine are reduced, and the shortening of a CIK cell culture cycle is more suitable for clinical treatment application.