Feruloyl glycerol (FG) has a variety of biological activities, but the green synthesis methods of FG remain rare. In this study, FG was prepared by a cascade reaction catalyzed by 4-coumarate coenzyme A ligase (4CL) and hydroxycinnamoyl acyltransferase 4 (HCT4). The cascade reaction carried out at solvent water and room temperature is more convenient and greener. Firstly, the product derived from the cascade reaction was characterized by TLC, HPLC, FTIR, and ESI-MS. The results showed that the product was FG. Secondly, the effects of temperature, pH, enzyme ratio, Mg2+ concentration, and CoA concentration on the cascade reaction were investigated. Consequently, the highest reaction rate was obtained at 30°C, pH 6, an enzyme ratio of 1:3, and Mg2+ concentration of 5mM. Finally, semi-preparative scale synthesis for FG was conducted. The production of FG reached 35.1mM at 24h with the FG conversion of 70.18%. In a word, a novel idea for the efficient and green synthesis of FG was proposed, which had great potential for industrial application.