In this study, the accumulation of γ-aminobutyric acid (GABA) in coffee leaves under ultrasonication and the impacts on total phenolic content (TPC), the activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), and angiotensinconverting enzyme (ACE) were investigated. The pH, incubation time, and ultrasonic power were the three most important factors that affected the accumulation of GABA. Coffee leaves treated with 20/40 kHz ultrasound have the highest GABA and the greatest GAD and ACE inhibitory activity. After optimization with a Box-Behnken design, GABA content was increased by 1.6 times under the optimal conditions of pH 5.9, incubation time 6.6 hr, and ultrasonic power 150 W. Our study indicated that ultrasonication activated GAD while inhibited GABA-T, thereby leading to an increase in GABA and ACE inhibitory activity. Ultrasonication also enhanced TPC; however, long time incubation caused the oxidation of phenolic compounds, and thus, affected the ACE inhibitory activity.Novelty impact statement: This is the first research that studied the enrichment of Gamma-aminobutyric acid (GABA) in coffee leaves using ultrasound stress. GABAenriched coffee leaves can be used to prepare tea-like beverage or natural health products to prevent hypertension, anxiety, and sleeplessness.