Natural killer (NK) cells-based immunotherapy is one of the most promising treatments for incurable malignant tumors. NK cells in combination with monoclonal antibodies to surface antigens of the tumor cell have been proved to be effective in a number of clinical trials. A limiting step in the development of successful cellular immunotherapy lies in developing an efficient and economic method to expand appropriate amount of NK cells and CD8(+) T cells. In this study, we constructed a humanized IL-15Ralpha-IgG1-Fc, which mimicked IL-15 trans-presentation. The feasibility of expanding populations of the human NK and CD8(+) T cells by IL-15Ralpha-IgG1-Fc complexes was tested. We then measured the cytotoxicity of expanded NK and CD8(+) T cells against tumor cell lines and primary tumor cells. When tested ex vivo, IL-2/IL-15Ralpha-IgG1-Fc complexes significantly enhanced NK and CD8(+) T cells expansion, isolated or non-isolated from PBMCs. The effect of IL-15Ralpha-IgG1-Fc was dependent on the presence of IL-2 or IL-15. IL-15Ralpha-IgG1-Fc complexes increased NK, CD8(+) T and NKT cells ratio in PBMC and BMMC after 14 days incubation. High level of granzyme B expression was observed in the supernatant of the complexes-treated NK cells. Expanded NK and CD8(+) T cell populations had cytotoxic function against the PC3, LNCaP, K562 and chronic lymphocytic leukemia (CLL) patient primary B cell lymphoma. We concluded that IL-2/IL-15Ralpha-IgG1-Fc significantly enhanced NK, CD8(+) T and NKT cells expansion, which possess strong anti-tumor activity. These data support clinical testing IL-2/IL-15Ralpha-IgG1-Fc expanded NK cells in patients with prostate cancer and CLL.