Objective To optimize the extraction process of alcohol-soluble proteins from highland barley fermentation spent, aiming to enhance the added value of byproducts from highland barley wine processing.Methods The alcohol-soluble proteins were extracted from highland barley fermentation spent using ultrasound-assisted ethanol extraction. The yield of alcohol-soluble proteins was used as the evaluation index to analyze the effects of four factors, i.e., solid-to-liquid ratio, ultrasound temperature, ultrasound power, and ultrasound time, on the protein yield. An orthogonal test was conducted to optimize the extraction process and determine the optimal extraction conditions. SDS-PAGE analysis was performed to examine the relative molecular mass distribution of the proteins and confirm their identity as alcohol-soluble proteins. Vitamin C (V_c) was used as a control to compare the antioxidant activities of the alcohol-soluble proteins, including DPPH· and ·OH radical scavenging rates and total reducing power.Results The optimal extraction conditions for alcohol-soluble proteins from highland barley fermentation spent were a solid-to-liquid ratio of 1:16 (g/mL), ultrasound temperature of 35 ℃, ultrasound power of 320 W, and extraction time of 60 minutes. Under these conditions, the yield of alcohol-soluble proteins was (18.50±0.020)%. SDS-PAGE showed that the extracted proteins had relative molecular masses mainly distributed between 11 kDa and 17 kDa and below 11 kDa, confirming the proteins as alcohol-soluble with low molecular mass. In vitro antioxidant assays indicated that the DPPH· and ·OH radical scavenging rates and total reducing power of the alcohol-soluble proteins increased with increasing protein concentration but were weaker than those of V_c at the same concentration. At a protein concentration of 10 mg/mL, the DPPH· and ·OH scavenging rates were (54.00±0.020)% and (22.90±0.010)%, respectively, and the Fe³⁺ reducing power reached its maximum absorbance of 0.50±0.100.Conclusion The alcohol-soluble proteins extracted from highland barley fermentation spent under the optimized conditions exhibited good in vitro antioxidant activity, indicating their potential for development as antioxidants.