3-Phenyllactic acid (PLA) is a natural product with broad and effective antimicrobial activity that is often used as a preservative in the food industry and as feed additive to replace antibiotics in livestock feeds. In this study, a two-step biocatalytic process for production of PLA from L-phenylalanine was developed and investigated. In the first step, L-phenylalanine was converted to phenylpyruvic acid (PPA) by recombinant Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. In the second step, unpurified PPA was directly converted to PLA by recombinant E. coli cells co-expressing D-2-hydroxyisocaproate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus and formate dehydrogenase from Mycobacterium vaccae N10. Through two-step cascade reactions, 121 mM PLA was produced from 150 mM L-phenylalanine with a yield of 81.3%. Thus, this process showed great production efficiency and is a promising method for industrial production of PLA.