Objective To establish in vitro isolation and culture technology of tree shrew retina-derived microvascular endothelial cells and immortalize cell lines to provide new experimental materials for in vitro study of tree shrew retinal microvascular endothelial cells. Methods Primary retinal microvascular endothelial cells were isolated by treatment with collagenase type Ⅱ, disperse and DNase I. The cells were purified by differential digestion. Morphological observation, immunofluorescence, and karyotyping were performed on cells passaged more than 50 times. Results Microvascular endothelial cells were isolated by enzyme digestion. Purified cells had an irregular polygonal shape and pavement pattern. The morphology of cells infected with a lentivirus was consistent after subculture, and cell morphology was intact at passage 50. Immunofluorescence showed that VWF, CD34, Claudin 1, ZO-1, and SV40T were positive. The growth curve showed that the cells grew vigorously and were in the logarithmic growth phase from day 2 to 4, which reached a plateau on day 4. Karyotyping showed that the chromosome number was consistent with the chromosome number of Sino-Burmese tree shrew. Therefore, the obtained cells were immortalized retinal microvascular endothelial cells. Conclusions The immortalized cell line of tree shrew retinal microvascular endothelial cells had an intact morphology, structure and functions, which provides new experimental material for the study of retinopathy and ophthalmology-related diseases.