In order to isolate the brain microvascular endothelial cells(BMECs) from Pelophylax nigromaculata, the brain tissue was used in this study. Under an asepic condition, the cerebral cortex was digested with 0.1% collagenase type II and collagenase/dispase, then separated by Percoll density gradient centrifugation. BMECs were inoculated in culture flask coated with rat tail glue, and cultured at 28 °C, 5% CO₂. Then, the cells were identified by morphologic observation and immunofluorescence staining with factor Ⅷ. Besides, the methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to detect the cell viability. The results showed that after 1 day, the microvascular fragments grew adherently and proliferated in a clustered monolayer, then expanded in culture and formed confluent monolayers with the characteristic cobblestone shape on 7th day. Positive Ⅷ expression was frequently observed in P. nigromaculata BMECs cytoplasm. Besides, MTT assay showed the cell viability of P. nigromaculata BMECs grew the fastest on the 3rd day to the 5th. In this study, an available method of culturing P. nigromaculata BMECs was established for the first time, which is of great significance for studying the pathogenesis of frog neurological diseases in vitro, providing a cell model for the development and screening of related drugs.