β-glucan extract (GE) was obtained from highland barley bran using alkaline–acid–alcohol extraction method. The stability, solubility, foaming ability, and prebiotics characteristics of GE were assessed consecutively. GE demonstrated excellent heat stability (hardly degraded at 220 °C) and pH stability, especially at neutral or alkaline condition, and its solubility was significantly influenced by temperature instead of pH or NaCl, achieving 0.91 g/100 g at 100 °C. Good foaming ability and foam stability of GE were observed during low temperatures (≤40 °C), neutral or alkaline condition. GE indicated a strong anti-digestibility capacity of resisting the hydrolysis of α-amylase and simulated human gastric acid. Interestingly, GE could effectively promote the growth of Lactobacillus bulgaricus and Bifidobacterium adolescentis, which was close to fructooligosaccharide. The results of this study could offer valuable information for the application of β-glucan from highland barley as prebiotics in promoting human intestinal health metabolism.