Andrographolide derivatives have been reported as potential α-glucosidase inhibitors. A more detailed investigation of active sites and key residues of α-glucosidase, which can interact with andrographolide derivatives, is useful for the discovery of more effective α-glucosidase inhibitors. To establish a virtual method predicting potential active sites and key residues, a 3D structure of α-glucosidase from baker's yeast was first constructed by homology modeling (RMSD of alignment was 1.745Å). The SiteID program was then used to explore the potential binding sites of the homology model of α-glucosidase. Based on these potential binding sites, 25 andrographolide derivatives were docked into the homology model of α-glucosidase for determining the potential active sites. Results showed that the predicted strong inhibitors' AR (accuracy rate) of the two potential active sites, P7 and P10, were 77.8% and 88.9% respectively. To further research the potential active sites, key residues of these predicted active sites were investigated through the virtual residues knockout method. VAL316 and THR307, residues of P7, and HIS258, LYS262, ILE271, MET272, THR273, ALA289, TYR292, and SER295, as well as residues of P10, were found to contribute greatly to the high ARs of their corresponding active sites. These results may be useful for design of new α-glucosidase inhibitors.