Natural α-amylase inhibitors (α-AIs) serve as food processing additives, capable of mitigating postprandial blood glucose levels, but heat resistance limits their application in high-temperature processing. This study delved into the correlation between protein structural characteristics and heat-resistance of colored highland barley (CHB) α-AIs and evaluated the inhibitory activity during chemical modification and in vitro digestion. Results demonstrated that CHB α-AIs were glycoproteins, the inhibitory activity retention rate of black highland barley α-AI salted-out with 0-60 % (NH