Objective To establish a stable and real-time monitorable nude mouse model of orthotopic glioma xenograft. Methods U251 glioma cell line was infected by a lentiviral vector containing green fluorescent protein (GFP) and luciferase (Luc) gene. Cells stably expressing fluorescence of GFP and Luc were sorted by flow cytometry. CCK-8 test and Transwell tumor invasion and migration assay were used to compare the biological features between the cells stably expressing GFP-Luc fluorescence and cells without fluorescence. Then the cells were implanted intracranially in the right caudate nucleus of athymic Balb/c nude mice to establish the tumor model. The growth of intracerebral tumor was monitored over time by a bioluminescence imaging (BLI) system. Hematoxylin-eosin (HE) staining was used to evaluate the histopathological features and tumorigenicity of the transplanted glioma cells in the brain of nude mice. Results U251 glioma cell line with stably expressing GFP-Luc fluorescence and the corresponding orthotopic xenograft model were successfully established. There was no statistically significant difference in the proliferation, invasion and migration abilities between the cells with stably expressing GFP-Luc fluorescence and the control cells. This model showed a high tumor formation rate and stable tumor growth, and takes a moderate time to establish this model. Conclusions Compared with the traditional glioma cells, GFP-Luc-transfected human glioma cells are more feasible for the studies of glioma in vivo. The tumor growth and pathological characteristics in this U251-GFP-Luc glioma model are similar to human glioma, and the growth of this tumor can be real-time monitored. It can be used as an ideal animal model for experimental studies of glioma.