Highland barley is a unique cereal in the Qinghai-Tibet Plateau of China, and has good effects on improving blood glucose metabolism. However, its specific hypoglycemic ingredients and compatibility are not yet clear. In this manuscript, the polyphenols, polysaccharides, and peptides in whole grain highland barley (WHB) were extracted. And the α-glucosidase inhibitory activity of 3 key active ingredients components and their interaction were investigated. The optimal reaction system for measuring α-glucosidase inhibitory activity using the microplate method was first determined through single factor experiments and Box-Benhnken response surface optimization experiments, i.e., 0.5 U/mL of enzyme concentration, 80 mmol/L of buffer concentration, and 5 mmol/L of substrate concentration. The results showed that all 3 WHB extracts exhibited good α-glucosidase inhibitory activity, with WHB polyphenols having the strongest inhibitory activity. Its inhibition rate reached 83.63% ± 2.92%, corresponding to an IC50 of 0.18 mg/mL. In addition, any 2 hypoglycemic ingredients combinations of the 3 WHB extracts had a certain synergistic effect. Our results will provide guidance for dietary choices in blood glucose control populations and new evidence for the development of functional foods.