As a new imaging technique, coherent Raman scattering (CRS) microscopy has been widely used in chemical structure and composition analysis because of its advantages of label-free, high specificity, and on-invasion. In recent years, the mutual cross-over and integrated development of photonics, biomedicine, and microscopic imaging technology has greatly promoted the application of CRS microscopy in biomedicine. This paper briefly introduces the basic principle of CRS microscopy and its classification, then explains the most widely used implementation of CRS, and summarizes the recent applications of CRS microscopic imaging in biomedicine, including detection, lipid analysis, and protein conformation change. Finally, the future development of CRS is discussed.