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.