Aim To establish an efficient and stable isolation method of primary mouse brain vascular smooth muscle cells and endothelial cells, and provide experimental materials for the investigation of pathogenesis and treatment of brain vascular diseases. Methods Brain vascular smooth muscle cells were isolated by dextran gradient centrifugation with enzymatic digestion, and endothelial cells were isolated by immunomagnetic beads sorting. Morphology and growth characteristics of two types of cells were observed with an inverted phase contrast microscope, their purity were identified by immunofluorescence, and their proliferation characteristics were observed by CCK-8 assay. At the level of cellular function, angiogenic capacity of endothelial cell was assessed by angiogenesis assay and smooth muscle cell responsiveness to platelet-derived growth factor (PDGF) was assessed by migration assay. Results The two types of cells isolated using this method grew vigorously and were in good condition. Smooth muscle cells exhibited typical “peak valley” growth, and immunofluorescence results showed cytoplasmic specific smooth muscle α-SMA and SM22α expression was positive. Endothelial cells exhibited typical “cobblestone like” growth, with positive expression of platelet endothelial cell adhesion molecule CD31 and atresia zone protein 1. Conclusion This study established a reliable and efficient method for simultaneously isolating two types of cerebrovascular cells, the isolated cells have high purity, good activity, and stable characteristics after passage, which were sufficient to meet the needs of subsequent experiments.