Activity guided-assisted high-speed counter-current chromatography (HSCCC) was used to separate α-glucosidase inhibitory components from blueberry in this study. Firstly， the active components were enriched in water after being extracted by different solvents. Secondly. the water extract was separated by HSCCC and six components were obtained. The inhibition rate of F4 on α-glucosidase was significantly higher than other components. Then F4 was purified by Sephadex LH-20 dextran gel to obtain high purity component I. Finally， component I was identified as cyanidin-3-O-glucoside (C3G) by UV， FTIR， HPLC-MS and NMR， and its purity was 95.06%. In addition， the interaction between C3G and α-glucosidase was characterized by multispectral scanning and molecular docking. It was found that C3G could spontaneously combine with α-glucosidase through hydrogen bond to form complex. After compounding with C3G， the secondary structure of α-glucosidase changed in varying degrees， in which the α-helix and β-angle decreased， while the β-folding and irregular helix increased. Molecular docking simulation showed that C3G was hydrogen bonded with Leu 313， Ser 157， Tyr 158， Phe 314， Arg 315 and Asp 307， and had hydrophobic interaction with four hydrophobic residues， which maintained the complex structure. The results of this study have important guiding significance for the development of functional foods for treating type 2 diabetes.