Natural killer (NK) cells are pivotal effector lymphocytes characterized for the innate immune response to pathogenic microorganism and tumor cells without priming and sensitization. Despite emerging knowledge has highlighted the rosy prospects in tumor immunosurveillance, yet the large-scale clinical application of NK cell-based therapy is hindered largely attributes to the defects in generating sufficient and high-quality cellular products. Herein, on the basis of 16 kinds of candidate combinations, we investigated the feasibility of cytokine cocktail-based strategy for convenient and standardized NK cell cultivation as well as the multifaceted characteristics and cytotoxicity against tumor cells. Our results revealed that joint utilization of Interleukin (IL)-2, IL-15, IL-18 manifested the optimal facilitation upon the ex vivo expansion and proportion of NK cells in peripheral blood mononuclear cells (PBMCs). Meanwhile, the obtained NK cell population expressed high levels of activating molecules (CD16 and NKG2D) and exhibited splendid cytotoxicity against K562 cell line. Collectively, with the aid of cytokine-based programming, we established an alternative strategy for facilitating the large-scale persistence and activation of NK cells from peripheral blood, which would benefit the NK cell- and chimeric antigen receptor-modified NK (CAR-NK) cell-based autologous or allogeneic tumor immunotherapy.