This study evaluated the impact of ultrasound pretreatment (UP) and enzyme type (flavourzyme, alcalase) on amino acid composition, antihypertensive, antidiabetic, and antioxidant activities of lupin protein hydrolysate (LPH). The UP altered molecular weight of the hydrolysates as revealed by SDS-PAGE and MALDI-TOF and their surface hydrophobicity and thermal stability. Amino acid analysis revealed that glutamic acid, aspartic acid, arginine, and leucine were the dominant amino acids lupin protein. Generally, sonicated hydrolysates exhibited higher biological activity than non-sonicated samples and the original LPI. Alcalase hydrolysate pretreated for 10 min showed the highest DPPH and metal chelating activities. Highest ACE inhibitory activity was observed in flavourzyme hydrolysate after 5 min of UP, while maximum α-amylase and glucosidase inhibitory activity was achieved after 10 min of UP in flavourzyme hydrolysate. The results obtained in this study demonstrated the potentials of UP as a valuable tool for enhancing the biological activity of lupin proteins.