ObjectiveTo explore the screening method for granulocytes with cancer killing activity (CKA) and to observe their in vitro antitumor effects. Methods(1) Anticoagulated peripheral whole blood samples were collected from 21 healthy volunteers and were cocultured with human lung cancer cell line A549.The proliferation activity of cancer cells was observed to screen granulocytes with CKA. (2) The screened granulocytes with CKA was used to treat various tumor cells, CCK8 method was used to examine the vitality of tumor cells, and the cell apoptosis was detected by Annexin ⅤFITC Apoptosis Detection Kit Ⅱ. (3) The cancer cells were cocultured using direct contact, Transwell and conditioned medium, and the effects of the three methods on the antitumor effect of CKA granulocytes were observed. Results(1) Six granulocyte clones showed noticeable inhibitory effect against A549 cells, and clone 3 had the most potent effect and was marked as CKA granulocyte. (2) The screened CKA granulocytes not only had noticeable inhibitory effects against A549, HepG2 and HeLa cells, but also promoted their apoptosis. (3)The order of the inhibitory efficiencies for the three culture methods from strong to weak was: direct contact > Transwell >conditioned medium. Conclusion CKA granulocytes have noticeable inhibitory effect against some cancer cells, which might be associated with their contact mode with the cancer cells.