Effect of ultrasound treatment on the physicochemical properties and structure of β‐lactoglobulin were investigated. β‐Lactoglobulin was treated with ultrasound at different amplitudes, temperatures, and durations. The surface hydrophobicity and free sulfhydryl group of β‐lactoglobulin were significantly increased after ultrasound treatment ( p < .05). The maximal surface hydrophobicity and free sulfhydryl group were 5,812.08 and 5.97 μmol/g, respectively. Ultrasound treatment changed the physicochemical properties of β‐lactoglobulin including particle size (from 1.21 ± 0.05 nm to 1.66 ± 0.03 nm), absolute zeta potential (from 15.47 ± 1.60 mV to 27.63 ± 3.30 mV), and solubility (from 84.66% to 95.17%). Ultrasound treatment increased α‐helix and β‐sheet structures of β‐lactoglobulin. Intrinsic fluorescence intensity of ultrasound‐treated β‐lactoglobulin was increased with shift of λ_max from 334 to 329 nm. UV absorption of β‐lactoglobulin was decreased with shift of λ_max from 288 to 285 nm after ultrasound treatment. There were no significant changes in high‐performance liquid chromatography and protein electrophoretic patterns. These findings indicated that ultrasound treatment had high potential in modifying the physiochemical and structural properties of β‐lactoglobulin for industrial applications.