Hyperspectral imaging method can obtain two-dimensional images and spectral information of in vivo tissues, which has the advantages of high spatial and spectral resolution, large imaging range, non-invasive nature, and fast speed, providing abundant information for in vivo tissue diagnosis. In recent years, researchers have made great progress in imaging methods, instruments, and applications. In this study, the main advances of hyperspectral imaging methods and applications are reviewed, and the spectral imaging methods, system composition, and characteristics are discussed. This study introduces the research progress in in vivo tissue imaging methods and application in terms of spectral reconstruction, tissue optical parameter measurement, and image processing based on deep learning. Simultaneously, the application progress of hyperspectral imaging in clinical medicine, such as skin trauma and healing process detection, diagnosis of diabetic foot and retinal diseases, intraoperative detection, and microcirculation function evaluation, is also summarized.