A novel method for enzymatic synthesis of L-theanine using copper（Ⅱ）-L-glutamine［Cu（Gln）2］as donor substrate was proposed.The structure of Cu（Gln）2 was identified by infrared spectrum analysis and its stability was also investigated under the reaction conditions.The enzymatic synthesis of L-theanine catalyzed by γ-glutamyltranspeptidase was carried out by using Cu（Gln）2 and L-glutamine as donor substrate respectively，and the product yield and conversion rate of donor substrate were compared under the conditions of different ratios of donor and acceptor.The results showed that the transpeptidation reaction could be effectively enhanced by using Cu（Gln）2 as donor substrate.The conversion rate of donor would increase by 51.5%，44.9% and 27.1% under different reaction conditions，compared to that using L-Gln as donor substrate.When the molar ratio of donor to acceptor was 6∶100，a higher donor conversion of 71.3% was obtained.