Abstract: Objective To investigate the effect of different stimulated objects, different stimulating concentrations, different protein transportation inhibitors on the frequency of human Th17 cells. Methods Whole blood or peripheral blood mononuclear cells （PBMC） were stimulated by PMA at different concentrations（25, 50, 150, 300, 500, 750, and 1 000ng/mL）. Ionomycin, different protein transportation inhibitors （BFA or Monensin） were added after 1h and then Th17 cells were detected after different stimulating time points （3, 6, and 9h）. Results The stimulating effect of whole blood was better than PBMC（P＜0.05）. The best final concentration of PMA stimulating the whole blood was 300ng/mL. BFA and Monensin had a similar inhibitory effect on IL-17 secretion, but influenced the process of hemolysis differently. Six hours were found to be more suitable to stimulate the whole blood. Conclusion Stimulating whole blood with PMA （300ng/mL） and Ionomycin, with 2μmol/L Monensin added after 1h and incubated for 6h, can detect the frequency of human Th17 cells.