Laser Raman spectroscopy is a physical probe that can be used to diagnose the molecular structure of tissues. In this study, Renishaw InVia laser confocal Raman spectrometer and 514 nm laser was used. A total of 14 cases of fresh grade Ⅲ and Ⅳ human brain glioma tissues and normal human brain tissue samples were measured. The results indicated that significant Raman peak-to-peak displacement at 1 664, 2 852, 2 882 and 2 930 cm–1 peaks were found between normal brain tissue and glioma lesion tissue. Furthermore, the difference in Raman peak-to-peak displacement between grade Ⅲ and grade Ⅳ glioma tissues were at 1 235, 1 260, 1 311, 1 360, 1 583, 1 660, 3 160 and 3 300 cm–1 peaks. Meanwhile, the differences of relative displacement at I1 660/I1 583、I1 311/I1 360、I1 235/I1 360 were obvious and showed diagnostic sense. In conclusion, Raman spectroscopy could distinguish brain tissues among normal brain, grade Ⅲ glioma and grade Ⅳ glioma, and the accuracy could be up to 86%. Thus, Raman spectroscopy could be used as an accurate and rapid method for glioma diagnosis and grading.