Effects of ultrasound (20–40% amplitudes at 45–55°C) and enzymatic (pepsin and trypsin) treatment on structure and antioxidant activity of β-lactoglobulin were studied. Changes in structure of β-lactoglobulin were investigated using spectroscopy techniques and changes in antioxidant activity were measured by chemical and cellular-based assays. Ultrasound treatment had considerable impact on the structure of β-lactoglobulin and increased the susceptibility of β-lactoglobulin to both pepsin and trypsin proteolysis. Intrinsic fluorescence intensity of β-lactoglobulin was increased by ultrasound and then decreased after following enzymatic treatment. Compared with control, the β-lactoglobulin after ultrasound and enzymatic treatments showed significantly higher oxygen scavenging activities in Caco-2 cells models, ABTS (2,2'-Azinobis-3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity and oxygen radical absorbance capacity (P < 0.05). Results indicated that ultrasound treatment increased the proteolysis of β-lactoglobulin by both pepsin and trypsin and improved the antioxidant activity of the protein and its proteolytic products.