OBJECTIVE : To evaluate the feasibility and clinical value of magnetic resonance diffusion tensor imaging (DTI) with fluorescein sodium staining (FLS) in the resection of high-grade glioma (HGG) in functional brain areas. METHODS : Retrospective cohort study design. The data of 95 patients who underwent surgery at the First Affiliated Hospital of Zhengzhou University for HGG in functional brain areas from October 2014 to December 2017 were investigated. In the observation group, 49 patients underwent DTI preoperatively and received FLS for the removal of tumor during the operation. In the control group, 46 patients received the routine method. All patients were subjected to enhanced magnetic resonance imaging to assess the extent of tumor resection within 72 hours after operation. The changes in muscle strength and Karnofsky Performance Status Scale (KPS) scores were evaluated 1 month after surgery. RESULTS : The extent of resection was significantly higher in the observation group than in the control group (83.7% vs. 45.7%, respectively; P < 0.001). The rate of muscle strength reduction after surgery was remarkably lower in the observation group than in the control group (20.4% vs. 47.8%, respectively; P = 0.005). KPS scores were higher in the observation group than in the control group (73.5% vs. 47.8%, respectively; P = 0.029). In the observation group, the sensitivity of FLS in identifying tumor tissue was 91.7% (44/48), with a specificity of 90.0% (45/50). CONCLUSIONS : The application of DTI with FLS can facilitate the maximum resection of HGG in functional brain areas with minimum loss of fiber tracts, reduce the disability rate, and improve quality of postoperative life compared with traditional glioma surgery.