Abstract: Angiotensin-Converting Enzyme Inhibitor(ACEI) peptides were prepared from double-low rapeseed protein by pulsed ultrasonic-assisted enzymolysis. Three reaction models, which included ultrasonic pretreatment of proteinase followed by enzymolysis (UPPE), ultrasonic pretreatment of double-low rapeseed protein solution followed by enzymolysis (UPRPE) and ultrasonic-assisted enzymolysis processes during previous period (UAE), were investigated. The inhibitory activity of hydrolysate to Angiotensin-Converting Enzyme(ACE) was the index. Traditional enzymatic hydrolysis (TE) without ultrasonic treatment was conducted as a control test. Results showed that three models all had significant effects, and UPRPE was the best. The optimum parameters of UPRPE obtained by orthogonal experiments were: ultrasonic frequency 20 kHz, ultrasonic treatment time 30 min, ultrasonic power 1250 W, on-time of the pulse 4 s and off-time 2 s. Compared with the traditional enzymatic hydrolysis, the inhibitory activity of the hydrolysate to ACE increased from 70.83% to 92.97%, ACEI peptide output raised from 29.6% to 41.4% and half inhibiting concentration IC50 value decreased from 3.57 mg/mL to 2.48 mg/mL under the optimum UPRPE conditions. These results prov that the pulsed ultrasonic-assisted enzymatic hydrolysis is a highly efficient method for preparing ACEI peptides from double-low rapeseed protein.