In order to improve the bioavailability of rapeseed meal protein concentrate (RPC), sequential dual frequency (50/28 kHz) ultrasound was applied to pretreat RPC and its protein bioavailability was investigated by in-vivo nitrogen-balance trials. Based on Box-Behnken experimental design, a set of preferred ultrasound pretreatment condition was determined to be at power density of 65.6 W/L for 38.6 min with an incubation temperature of 39.0 degrees C. Under these conditions, degree of hydrolysis and protein conversion rate of RPC respectively increased by 26.6 % and 32.9 % compared to the control. Meanwhile, the macromolecular protein of sonicated RPC was converted into more small molecular peptides with a molecular weight of 200-1000 Da by enzymolysis, accompanied by the changes of amino acid composition in supernatant. Furthermore, the rats fed with hydrolysates (including supernatant and precipitate) derived from ultrasound pretreatment coupled with enzymolysis rather than traditional enzymolysis showed higher weight gain, protein efficiency ratio, net protein ratio, apparent digestibility and true digestibility. These findings revealed that ultrasound could be considered as a promising pretreatment technology for preparing enzymatic hydrolysates with high bioavailability and thus realize the maximum utilization of rapeseed meal protein.