Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of l-phenylglycine (l-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD(+)-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs (4) leudh-3fdh (A10C) was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs (4) leudh-3fdh (A10C) was 28.4 mg L-1 min(-1) g(-1) dry cell weight for l-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 A degrees C), 60 g L-1 benzoylformic acid was completely converted to pure chiral l-Phg in 4.5 h with 10 g L-1 dry cells and 50.4 g L-1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved l-Phg productivity and demonstrated a novel low-cost method for enantiopure l-Phg production.