Objective The aim of this study was to develop and assess a genetically engineered mouse model for visualizing in vivo fluorescence of glioma, mural cells, and blood vessels using two-photon microscopy. Methods PDGFRβ-Cre(+/-):Rosa26-tdTomato(+/-) genetically engineered mice were underwent skull clearance and injected with GL261-CFP. This was done to study the dynamic alterations in blood vessels and mural cells during the progression and invasion of glioma using a two-photon microscope. Results PDGFRβ-Cre(+/-):Rosa26-tdTomato(+/-) mice were successfully bred and subjected to HE section analysis of functional organ tissues, revealing no discernible differences from C57BL/6 mice in terms of appearance and morphology. Cre recombinase activity was fully induced following Tamoxifen treatment on day 7. Subsequent GL261-CFP inoculation demonstrated the dynamic progression of glioma proliferation and invasion, as well as vascular abnormalities and increased mural cell detachment within the tumor. Conclusions The genetically engineered mice that mural cell expressing fluorescent were bred successfully. Utilizing fluorescein isothiocyanate-dextran labeling of blood vessels and blue fluorescent tumor cells, glass discs and fixed rings were employed to replace the skull of the mice. This allowed for the tracking of morphological and structural changes in blood vessels and vascular supporting cells following the development of brain tumors in vivo over an extended period. This model offers a valuable tool for studying brain diseases through pathological visualization.