Hyperspectral imaging is an emerging technology for various medical applications, especially in disease diagnosis of early and biomedical research based on their unique spectral signatures. A hyperspectral microscopy based on a prism-grating-prism is designed for various medical applications. The spectral range between 400 nm to 800 nm is sampled by 256 spectral channels, the modulation transfer function (MTF) values of full spectrum and full field of view are close to the diffraction limit, the smile is less than 6.4% of a pixel, and the keystone is less than 1.6% of a pixel. At the same time, the hyperspectral microscopy used an internal pushbroom technology, which has the advantages of compact structure and good portability. The spectral imaging results demonstrate the great potential of this internal pushbroom hyperspectral microscopy for the pathologists to automatically detect tumor tissue in pathological slides and early disease diagnosis.