Autophagy plays a critical role in many vitally important physiological and pathological processes, such as the removal of damaged and aged organelles and redundant proteins. Although autophagy is mainly a protective process for cells, it can also cause cell death. In this study, we employed in situ and ex situ surface-enhanced Raman scattering (SERS) spectroscopies to obtain chemical information of lysosomes of HepG2 cells. Results reveal that the SERS profiles of the isolated lysosomes are different from the in situ spectra, indicating that lysosomes lie in different microenvironments in these two cases. We further investigated the molecular changes of isolated lysosomes according to the autophagy induced by starvation via ex situ SERS. During autophagy, the conformation of proteins and the structures of lipids have been affected, and autophagy-related molecular evidence is given for the first time in the living lysosomes. We expect that this study will provide a reference for understanding the cell autophagy mechanism.