Objective：To investigate the effect of in vitro time and anticoagulant concentration on platelet activation. Methods：The pe－ripheral blood platelet of 85 healthy adults was exposed to different in vitro time and different concentrations of sodium citrate，then flow cytometry（FCM） and monoclonal antibodies（McAb） were used to detect the expression levels of platelet surface glycoprotein protein PAC-1，CD62p，CD63，thrombin-sensitive protein（TSP）. Results：Under the same in vitro time，platelet activation was in－creased with the concentration of sodium citrate increasing from 0.109 mol/L to 0.872 mol/L. Platelet activation did not change signif－icantly when the citrate concentration was higher than 0.872 mol/L. Platelet activation was slight increased within 2 h in vitro，signif－icantly increased within 2-8 h，remained unchanged after 8 h. Conclusions：Increasing the anticoagulant concentration within certain range and extending time in vitro can enhance platelet activation. We should choose the most appropriate anticoagulant concentration and the shortest time in vitro to test the platelet-related indicators and select appropriate storage method to save the platelet in order to reduce its activation.