<p>This study aimed to assess the primary phenolic groups in black highland barley (BHB) polyphenols that exhibit in vitro antioxidant and hypoglycemic activities, providing guidance for their effective utilization. BHB polyphenolic extracts were fractionated using petroleum ether, ethyl acetate, n-butanol, and water, with each fraction analyzed for phenolic and flavonoid content, polyphenolic composition, DPPH· and ABTS⁺· radical scavenging activity, FRAP reducing ability, and inhibitory effects on α-amylase and α-glucosidase. Among these, the n-butanol fraction demonstrated the highest total phenolic (2047.42&#xa0;μg/g), total flavonoid (161.73 µg/g), and total anthocyanin content (1.85 µg/g), along with the strongest antioxidant capacity. The ethyl acetate fraction excelled in inhibiting α-amylase (IC₅₀ = 60.38&#xa0;mg/mL) and α-glucosidase (IC₅₀ = 9.83&#xa0;mg/mL), followed by the n-butanol fraction. Additionally, the water fraction also displayed relatively high antioxidant properties. A total of 273 phenolic compounds were qualitatively identified, with flavonoids (171 compounds) as the predominant group. Ethyl acetate was more effective in enriching flavonoids, while water preferentially enriched phenols. Catechin was the most abundant compound across organic fractions (0.668–258.017&#xa0;μg/g), while benzoic acid was predominant in the water fraction (0.394&#xa0;μg/g). A clear correlation was noted between major polyphenolic compounds and in vitro bioactivities, notably chlorogenic acid, diosmin, kaempferol-3-O-rutinoside, epicatechin, and diosmetin. Overall, the n-butanol fraction, rich in bioactive polyphenols, has a predominant advantage in exerting both antioxidant and hypoglycemic activities in vitro, whereas the ethyl acetate and water fractions are advantageous in hypoglycemic and antioxidant activities, respectively.</p>