Protein conformational dynamics, often associated with static and dynamic inhomogeneities, plays a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensemble-averaged measurement, especially when the protein involves in a multiple-step complex chemical reaction, such as an enzymatic reaction. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics in real time under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. In this minireview, we discuss our recent studies on single-molecule enzymatic reaction dynamics and protein conformational dynamics of the T4 lysozyme hydrolyzation reaction of a polysaccharide by a combined approach of single-molecule spectroscopy, molecular dynamics simulation, and theoretical modeling. Detailed characterization of the complex enzymatic reaction dynamics identified the nature of the inhomogeneity and revealed multiple intermediate conformational states associated with the enzyme-substrate complex formation in the multiple-step enzymatic reaction.