The γ–Glutamyltranspeptidase gene from Escherichia coli DH5α was cloned in pET–32α vector and expressed in Escherichia coli BL21. When the recombinant strain grew at pH 7 to obtain the OD600 nm of 1.5 and induced with 0.1 mmol/L IPTG at 20 ℃ for 6 h, the activity of γ–Glutamyltranspeptidase was (4.41±0.17) U/mL, about 18.4 times than that of Escherichia coli DH5α. Under the catalysis of cells of the genetically engineered strain and under the pH 10, the temperature of 20 ℃ and the incubation time of 6 h, the yield of theanine from L–Gin (200 mmol/L) and ethylamine (1.5 mol/L) was 12.6 mg/mL, and the rate of conversion from L–glutamine to theanine was 41.05%.