Background Cord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines. Results The large numbers (mean: 1.59 x 10(10)) of highly pure (>= 90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-gamma, TNF-alpha and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells. Conclusion We develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.