To realize the high-value utilization of buckwheat protein resources， α-glucosidase-inhibiting glycopeptide (α-GIG) was prepared by fermentation of Lactobacillus plantarum and Streptococcus thermophilus which were selected from 6 strains. One-factor-at-a-time method aimed to determine the level of experimental factors and a further response surface experiment was used to optimize fermentation. The fermentation broth was separated and purified by Sephadex G-25 and reversed phase-high performance liquid chromatography (RP-HPLC) to prepare α-GIG. The method of β-elimination reaction was used to determine the type of glycopeptide bond and infrared spectroscopy was used to analyze the secondary structure. The inhibitory effects of α-GIG on α-glucosidase was investigated by enzyme kinetics. The results showed that when the inoculum was 2%， pH was 7.5， solid-liquid ratio was 1∶14.8 and fermentation time was 2.8 d， the α-glucosidase inhibition rate was 70.83%. I1， I2， I3 were separated from Sephadex G-25， and I2 with the best enzyme inhibitory effect whose IC50 is 1.72 mg/mL. I2 was further separated and purified by RP-HPLC to obtain α-GIG with a purity of 94.17%. Fourier transform infrared spectroscopy suggest that the secondary structure composition was estimated as 70.51% β-folding， 18.95% α-helices and 10.54% β-corner. β-elimination reaction demonstrated the existence of O-glycosidic linkage in α-GIG. The results of the enzyme inhibition kinetics study showed that α-GIG exhibited a mixed-type noncompetitive inhibition of α-glucosidase. Therefore， α-GIG had the potential to be developed as a natural α-glucosidase inhibitor.