In enzymology, a theanine hydrolase (EC 3.5.1.65) is an enzyme that catalyzes the chemical reaction

N⁵-ethyl-L-glutamine + H₂O ${\displaystyle \rightleftharpoons }$ L-glutamate + ethylamine

Thus, the two substrates of this enzyme are N⁵-ethyl-L-glutamine and H₂O, whereas its two products are L-glutamate and ethylamine.

This enzyme belongs to the family of hydrolases, those acting on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides. The systematic name of this enzyme class is N5-ethyl-L-glutamine amidohydrolase. Other names in common use include L-theanine amidohydrolase, and 5-N-ethyl-L-glutamine amidohydrolase.

• Tsushida T, Takeo T (1985). "An enzyme hydrolyzing L-theanine in tea leaves". Agric. Biol. Chem. 49 (10): 2913–2917. doi:10.1271/bbb1961.49.2913.