Objective To establish an efficient method of NK cell expansion and culture in vitro. The effectiveness of NK cells in killing three prostate cancer (PCa) cell lines was tested to evaluate its feasibility in the later treatment of PCa, which laid a foundation for the application of adoptive NK cell transfusion for PCa. Methods The NK cells were cultured by inducing and amplifying PBMCs with cytokines, and the proportion and amplification of NK cells were detected by flow cytometry. The efficiency and effect-target ratio of NK cells against three prostate cancer cell lines PC3, LNCaP and DU145 were measured by CCK8 method. Results The NK cells were successfully amplified. After PBMCs induction, NK cells were amplified from the initial 10.97%±3.28% to more than 83.20%±8.54%, and the total number of cells was (251.66±19.05) times. The amplification factor of NK cells was (1940.17±402.22). The killing rate of NK cells to the three kinds of target cells was significantly different (P＜0.01). The killing efficiency of NK cells against LNCaP was greater than that against DU145. It has no killing effect on PC3, but promotes its growth. The highest killing efficiency of NK cells against prostate cancer cells were LNCaP 92.03%±9.95%, DU145 72.40%±8.30% and PC3 26.69%±10.56%, respectively.Conclusion The proliferation of PBMCs induced by cytokines can obtain high purity and sufficient number of NK cells, which lays a foundation for the clinical application of NK cells. The killing rate of NK cells to the three prostate cancer cells was significantly different, which provided a direction for further research to explore the causes and mechanism of action