The invention discloses an enzymatic preparation method of L-theanine by utilizing L-glutamine-Copper (II) complex as donor, which comprises that adopting chemical process to synthesize L-glutamine-Copper (II) complex and Gamma-glutamyltranspeptidase is added into the mixed aqueous solution of containing substrate of L-glutamine-Copper (II) complex and ethylamine for reaction. The method has the advantages of avoiding auto-transpeptidation problem, greatly promoting the conversion rate of donor, realizing the simple operation and the mild reaction condition as well as the low cost.