Abstract: Angiotensin-converting enzyme (ACE) plays an important role in blood pressure regulation, and inhibition of its activity is beneficial to maintain blood pressure balance. Food-derived ACE inhibitory peptides have attracted wide attention due to their safety and easy absorption. This study aimed to obtain a new peptide of high ACE inhibitory activity from tea residue protein. With the ACE inhibitory rate of ACE inhibitory peptides as the index, ultrasonic pre-treatment of tea residue protein was the best method through comparison of three ultrasonic methods. Response surface optimization was used to determine the optimal ultrasonic pretreatment parameters based on the single-factor tests. The enzymatic solution was separated and purified by ultrafiltration, and the stability of components with molecular weight less than 3 kDa was analyzed. The results showed that the optimum ultrasonic pretreatment conditions were ultrasonic power 300 W, ultrasonic temperature 45 °C, ultrasonic time 25 min. Under the optimal conditions, the ACE inhibitory activity was 64.8%, which was 10.7% higher than 54.1% in the control group (without ultrasonic). When the molecular weight cut-off was less than 3 kDa, the inhibition rate of ACE inhibitory peptide was 82.3%, which was 17.5% higher than that of the original hydrolysate. When the temperature rose from 30 to 90 ^oC, the inhibition rate of ACE inhibitory peptide decreased from 82.3% to 78.3%. The peptide showed stable ACE inhibition rate in pH and salt solution. The inhibition rate of ACE inhibitory peptide decreased from 82.3% to 62.3% after 8 h in simulated digestion environment.