Abstract: Objective:To establish a thymosin activity detection method using Jurkat cell proliferation assay, and to optimize and verify it.Methods:Jurkat cells were cultured and stimulated with different concentrations of thymosin for 24 h or 48 h, and then the proliferation level of Jurkat cells was detected by cell counting kit-8(CCK-8) assay.Meanwhile, the specificity, repeatability, sensitivity, precision and applicability of Jurkat cell proliferation method to detect thymosin activity were verified.Results:The optimal detection conditions were when the concentration range of thymosin was 20-200 μg/mL, the stimulation time was 24 h and the incubation time of CCK-8 solution was 3 h.The activity of thymosin was in the range of 20-200 μg/ mL, and the relative increase rate of proliferation showed a dependence of concentration; all the RSDs for repeatability verification were less than 15%; the activity of thymosin after disruption detected by Jurkat cell method significantly decreased, and was positively correlated with the thymosin activity results detected by E-rosette method(r=0.75, P< 0.05).Conclusion:The thymosin activity detection method based on Jurkat cell proliferation assay has the advantages of accuracy, objectivity, sensitivity and stability, which can meet the requirements of thymosin activity detection and can be further popularized.