The inhibitory effects of L-theanine on the activities of α-amylase and trypsin were studied by establishing the best reaction system and adopting research method on graph in order to provide reference for the deep development of L-theanine. The results showed that the inhibitory effects of L-theanine on both enzyme activities were positively associated with its concentration. IC₅₀ (26.078 mg/mL) of L-theanine on the activities of α-amylase was achieved by prewarming enzyme at a concentration of 0.018 mg/mL in the presence of L-theanine at 37 ℃ for 5 min before addition of 1% starch and allowing reaction to proceed for 3 min. Km of α-amylase was 2.247 mg/mL on the premise that the enzyme amount was 18 518.52 U. L-theanine exhibited the half inhibition on trypsin (196.299 mg/mL) by prewarming their mixture at 40 ℃ for 5 min followed by addition of 1% casein as the substrate and permitting the reaction for 15 min. When the amount of enzyme was 1 055 931 U, Km of trypsin was 5.189 mg/mL. Statistics results showed that the inhibitory effect of L-theanine on the activity of α-amylase was superior than that of trypsin.