Objective：To Establish the methods of extracting and cultivate human aortic endothelial cells（HAECs）in vitro. Methods：Stripping endodermis from human aorta，using collagenaseⅠto digest endothelial cells from endodermis，and then utilizing immunohistochemistry and flow cytometry（FCM）to detect purity of HAECs. Results：0.1% and 0.2% collagenase type Ⅰ，the best digestion time was 50 min，the adherent cells number in 2 h were （25.2 ± 2.1）×103，（40.5 ± 3.8）×103. After cell fusion，cells were cobblestone-like growth in inverted microscope. Cells were identified by CD31 and Ⅷ factors as positive，the cell purity reached （96.43 ± 4.12）%. The ability of endocytosis of ox-LDL and angiogenesis were normal. Conclusion：The method that blunt dissection of the aortic endothelium and simply use type Ⅰ collagenase digestion was better to separate HAECs. Enzyme concentration and digestion time are important factors. The method is simple and convenient，shortening the training cycle and obtaining high purity cells. It is beneficial to the endothelial cells experiment in vitro.