Hyperspectral imaging (HSI) is a new rapid and safe modern medical detection technology. In this paper, a self-developed hyperspectral imager was combined with a microscope to study the imaging spectrum of glioma tissue in human glioma mice and H&E staining pathology slides. The hyperspectral data of glioma and normal tissues were analyzed and processed to find the difference between them so as to realize the distinction of them. The experimental results show that there are some differences in the reflectivity within a specific spectral range between glioma and normal tissues, whether in pathology slides or in vivo data. We can distinguish glioma and normal tissues and provide reference and gist for clinical trials and auxiliary diagnosis in glioma surgery base on these differences.