The invention relates to an active factor for enhancing lymphocyte targeting killing tumors and a preparation method thereof, wherein the amino acid sequence of the active factor is showed as SEQIDNO: 1. The preparation method comprises the following steps of: respectively amplifying the sections of SEQIDNO:2-4 by utilizing PCR (Polymerase Chain Reaction), inserting the three sections into a PQ31 vector in series, and connecting flexible sections among all sections to obtain a fusion protein expression vector; transforming the fusion protein expression vector into an M15 escherichia coli strain, carrying out IPTG inductive expression, then washing an inclusion body, purifying by using Ni+-ITA resins under the condition of denaturation, and further renaturing to obtain a dsNKg2D-IL-15 protein. The preparation method can express a large number of dsNKg2D-il-15 proteins in vitro, and the expressed protein is stable and not degradable in physiological environment, and has the ability of ensuring that tumor cells are in anti-form presentation on IL-15 to activate the activities of NK cells.