Cellular physical microenvironment such as crowding shows great influence on enzymatic reactions. Herein, we report a new finding that saccharides with low molecular weight create an effective crowding microenvironment for gene expression in cell-free protein synthesis, which provides valuable implications for living systems. Four saccharides including sorbose, galactose, sucrose, and cellobiose are screened out as effective crowders. At a low concentration range of saccharides, both the mRNA and protein amounts present an upward trend with the concentration increment of saccharides; when the concentrations exceed a critical value, the mRNA and protein amounts decrease. A mechanism is proposed that at low concentrations of saccharides, the effective concentrations of reactants increase due to the coexistence of crowders and reactants in a finite volume; when the concentrations exceed a critical value, the molecular diffusion of reactants is dominantly restricted due to the increased viscosity. Our finding opens a new view that saccharides with low molecular weight could be crowders and provides a new insight that substances with low molecular weight in cells would produce a crowding effect on biochemical reactions in living systems.