Abstract: Highland barley distillers' grains (HBDG), a major by-product of highland barley wine production, contain β-glucan as one of its principal bioactive components. To promote resource efficiency and fully exploit the potential value of HBDG, this study utilized HBDG as raw material to optimize the extraction process of β-glucan using ultrasound-assisted solvent extraction and systematically evaluated its effects as a functional additive on yogurt quality. Initially, the effects of ultrasonic power, ultrasonic time, extraction temperature, pH, and liquid-to-solid ratio on β-glucan extraction yield were systematically investigated to determine the optimal extraction parameters, with extraction yield serving as the indicator. Subsequently, the extracted β-glucan was incorporated into fermented milk at concentrations ranging from 0 to 1.0% (w/v). Its effects on yogurt acidity, pH, water-holding capacity (WHC), textural properties, viscosity, rheological characteristics, microstructure, and sensory attributes were determined. The results indicated that the optimal extraction conditions for β-glucan from HBDG were: ultrasonic power 256 W, ultrasonic time 10 min, extraction temperature 75 °C, pH 9.1, and liquid-to-solid ratio 10:1 mL/g. Under these conditions, the extraction yield reached 50.76%±0.63%. Regarding yogurt quality, increasing the β-glucan addition level significantly increased the pH, WHC, and viscosity(P<0.05), while significantly decreasing the acidity(P<0.05). Notably, yogurt supplemented with 0.5% (w/v) β-glucan exhibited superior textural properties and higher storage modulus (G') and loss modulus (G''), indicating improved viscoelasticity. This formulation also achieved the highest sensory score. Scanning electron microscopy (SEM) revealed that β-glucan addition promoted aggregation between casein micelles. In conclusion, ultrasound-assisted solvent extraction is an efficient method for isolating β-glucan from HBDG. Incorporating this β-glucan into yogurt at a concentration of 0.5% effectively enhances product texture, rheological properties, and microstructure while improving sensory quality. These findings provide a theoretical foundation and technical pathway for the high-value utilization of HBDG resources and the development of functional dairy products.