To understand molecular mechanism of biological functions, chemical reactions of biological molecules should be elucidated. For that purpose, we have developed new techniques to reveal the spectrally silent dynamics of the reactions and to unify the thermodynamics and kinetics of the reactions. In particular, we focus our attention on the “fluctuation” in this review. For example, proteins are flexible systems and the structures should be fluctuating at room temperature in aqueous solution due to the thermal energy. We found that the “fluctuations” of biological molecules are key factors for understanding reactions, and essentially important for understanding the functions. For proving the importance of the fluctuation, we showed that the fluctuation is indeed enhanced during the chemical reactions of biomolecules. We mainly used the transient grating (TG) method to detect the time dependence of the thermodynamical properties and diffusion coefficients. In particular, we succeeded in measurements of the thermal expansion coefficient, the heat capacity, and the isothermal compressibility, which are directly linked to the fluctuation, in time domain. These techniques have quite wide applicability to many reaction systems. We found that the fluctuation is closely related with the high efficiency and selectivity of the reactions of proteins.