Objective:To culture and identify endothelial cells from human umbilical vein endothelial cells(HUVECs) in vitro. Methods:HUVECs were obtained using 0.1% collagenase typeⅡ by the technique of irrigative digestion, and were cultured with M199 which contained fetal calf serum in the carbon dioxide incubator,and then were passaged using tryple express digestion. The culture cells were identified by morphology and flow cytometry detecting CD34. Results:HUVECs could adhere to the plate completely after 24 hours and got a monolayer 3~5 days later. The culture cells were HUVECs confirmed by morphology and flow cytometry. Conclusion:Using collagenase typeⅡand tryple express could obtain a large number of HUVECs in continuous passage culture.