An understanding of the thermodynamic properties of elementary chemical steps of a reaction is important for the development of fundamental reaction theories and for effective industrial practice. In this work, temperature-variable single-molecule fluorescence microscopy was employed to study a reversible redox chemical process and reveal the thermodynamics of chemical elementary reactions at a single-molecule level. Activation energies of pure elementary steps were measured on the level of single molecules and found to be heterogeneously distributed across the population of individual molecules. The activation parameters measured across the population of individual molecules also exhibited a compensation effect and an isokinetic relationship. These results constitute a new single-molecule-level perspective into a chemical reaction.