This study examined the effects of ultrasonic power (0, 120, 360, and 600 W) for 30 min on the structural, physicochemical and emulsifying properties of whey protein isolate (WPI) under different pH values (3, 5, 7, 9 and 11). The particle size distributions of WPI narrowed as the ultrasonic power increased from 0 to 600 W. WPI showed the maximum surface hydrophobicity and fluorescence intensity at 600 W and pH 11. Ultrasound significantly reduced the WPI turbidity at pH 3, 7, 9 and 11 and improved the WPI emulsion activity and stability at pH 3, 5, 7 and 9. Moreover, the number of beta-sheets and random coils dramatically increased in WPI at 600 W of ultrasonic power and pH 3, 7, 9 and 11. Overall, the combination of ultrasound and alkaline conditions could potentially alter the secondary structure of WPI and enhance its emulsifying properties.