Polyphosphate kinase (PPK) and γ-glutamylmethylamide synthetase (GMAS) were recombinant expressed using pETDuet-1 plasmid in Escherichia coli BL21(DE3), and L-theanine was synthesized by whole cell catalysis with co-expression of PPK and GMAS. The parameters of reaction were investigated and optimized. The SDS-PAGE analysis showed that PPK and GMAS were successfully co-expressed in the recombinant strains. The optimal conditions for whole cell biocatalytic reaction were as follows: 35 oC, pH=7.0, 300 mmol/L monosodium glutamate, 420 mmol/L ethylamine hydrochloride, 100 mmol/L polyP, 2 mmol/L ATP. After 24 h in a 100 mL reaction system, under the optimal conditions the L-theanine yield can reach 199 mmol/L, and the conversion rate reaches 66.34%.