Protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneities, play a crucial role in biomolecular functions. It is extremely difficult to characterize such spatially and temporally inhomogeneous dynamics in an ensemble-averaged measurement, especially when the proteins involve in a multiple-step and multiple-conformation complex chemical interactions and transformations, such as in protein-protein interactions and protein- DNA interactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structurefunction mechanisms.