There are a variety of ways for isolation and selective cultivation of retinal microvascular endothelial cells in vitro, in which the newly reported tissue culture with collagenase digestion method are widely used for simple, economical, high purity and good activity of obtained endothelial cells. As a result of the limitation of person eye donor, researchers often use rat retinal microvascular endothelial cells that are highly homologous to human beings to experiment in vitro. In this paper, the methods of culturing retinal microvascular endothelial cells in vitro will be elaborated from the aspects of animal species, added factors, cell purification and identification in vitro. At the same time, the advantages and disadvantages of these methods in the process of operation are briefly described, which can provide a reference for the researchers to select suitable cultivation methods.