Objective：To establish a method to isolate and culture regulatory T cells in vitro with high efficiency. Methods：CD4+CD25+CD127low T cells were isolated by fluorescence-activated cells sorting and cultured in vitro with anti-CD3-anti-CD28-coated microbeads and IL-2 in 2 weeks. Count cells numbers to evaluate the expansion ability，phenotype of expanded cells were identified with flow cytometry and the suppressive function was examined by mixed lymphocyte reaction. Results：A mean expansion of （755.5 ± 213.5） fold was obtained after cultured for 2 weeks. In addition，the expression of CD4，CD19，CD8，and CD25 in the expanded cells are（98.3 ± 1.04）%，（0.039 ± 0.021）%，（0.443 ± 0.239）% and（97.6 ± 1.35）% respectively. FOXP3+ cells accounts for（87.7 ± 5.5）% and Helios+ cells accounts for （73.3 ± 2.9）% of expanded cells. Cultured cells shows highest suppressive capacity with（84.39 ± 1.98）% suppression of activated PBMC at responder∶Treg ratio of 1∶1. Conclusion：The research provides a method to obtain large quantity of Tregs with high efficiency in vitro. The expanded cells remain their original phenotype and have ability of immune suppression which has great significance in cell therapy for treating autoimmune diseases and transplant rejections.