Adoptive transfer of NK cells has been widely applied clinically for cancer immunotherapy. However, the difficulties to obtain a large number of activated NK cells impede the successful application of such therapy. In the present study, we implemented a novel method involving the use of immobilized human 4-1BBL and interleukin-21 to amplify NK cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors. Following stimulation for 21 days, we achieved considerable expansion of NK cells with high purity and strong cytotoxicity. This is the first time solid phase cytokines were used to augment NK cells, and this method has the advantage of no need to introduce feeder cells, without prior purification of NK cells and it effectively stimulated and expanded NK cells. The strategy of cell proliferation and activation could lead to a safer and more effective application of NK cells clinically.