[Objective] L-homophenylalanine (L-HPA) is a key intermediate for many pharmaceuticals and pesticides. As chemical synthesis of L-HPA was complicated and environmentally unfriendly, this study aimed to develop an efficient and environmentally friendly route for the biosynthesis of L-HPA.[Methods] A multi-enzyme cascade reaction for L-HPA production from L-glycine and phenylacetaldehyde was constructed by modular assembly method.[Results] Firstly, the multi-enzyme cascade reaction, which consisted of threonine aldolases (TA), threonine deaminase (TD), phenylalanine dehydrogenase (PheDH), and formate dehydrogenase (FDH), was constructed by literature mining. Then, according to the introduction and deletion of chiral -OH/-NH₂ groups, the whole cascade reaction was divided into two parts, in which the basic parts included TA and TD and the extender parts included PheDH and FDH. Next, the two modules were assembled and optimized with different plasmids, enhancing the L-HPA production up to 208.6 mg/L in the optimal engineered strain BL21-C-M1-R-M2. Finally, the whole cell catalytic system was optimized to improve the L-HPA production up to 1226.6 mg/L and the molar conversion of phenylacetaldehyde reached 34.2%.[Conclusion] This environmental-friendly and efficient process represented a promising strategy for large-scale L-HPA production in the future.