Objective To explore the effect of magnetic resonance spectroscopy (MRS) combined with sodium fluorescein (FL) navigation in patients with supratentorial high-grade gliomas. Methods 94 patients with supratentorial high-grade gliomas were randomly divided into control group (routine microsurgery) and observation group (MRS + FL navigation surgery), 47 patients in each group and the treatment efficacy of two groups were compared. Results The total tumor resection rate of the observation group was 95.74%, which was higher than that of the control group (72.34%). The KPS score and tumor progression free survival time of the observation group were higher (longer) than those of the control group (P< 0.05). Conclusion MRS + FL navigation surgery is effective, optimizes the quality of life and prolongs the progression free survival time of tumor.