This study investigated the impact of ultrasound treatment on the physicochemical properties and stability of β-glucan-enriched highland barley (HB) milk. Ultrasound treatment significantly improved the physical stability of the HB milk, as evidenced by an increased storage index (SI) and the absolute value of the zeta potential after 8 days of storage. The reduced ΔBS value indicated the separation of the supernatant is visibly decreased, and the particle size distribution became narrower and more uniform. UV and fluorescence spectroscopy results proved that ultrasonic increased the hydrophobicity of the proteins by unfolding the molecular structure. Fourier transform infrared (FTIR) spectroscopy analysis revealed changes in the secondary structure of HB proteins, with a decrease in the proportion of α-helix and an increase of β-sheet and random coil. Confocal laser scanning microscope (CLSM) demonstrated that ultrasound treatment altered the distribution of proteins and β-glucan, promoting molecular interaction and enhancing stability. Combining color and turbidity assessments, ultrasound shows promising effect for enhancing the stability of HB milk.