In this study, the fed-batch fermentation technique was applied to improve the yield of α-amylase inhibitors produced by S.coelicoflavus ZG0656. Various fermentation substrates and conditions were investigated to identify the optimal concentration of carbon source in the production of α-amylase inhibitors. The ratio of maltose and glucose were found to be the optimal initial carbon source, and the optimal ratio containing 3:1 (mass ratio) of maltose and glucose and its optimal concentration was determined to be 100 g/L based on the results of fermentations conducted in a 5-L jar fermenter using a series of fed-batch cultures of S.coelicoflavus ZG0656. The effects of mixed carbon sources concentration and medium osmolality on the production of α-amylase inhibitors were also investigated in this work. Our results showed that the production of α-amylase inhibitors by S.coelicoflavus ZG0656 was enhanced when the feeding medium containing 2:1(mass ratio) of maltose and soluble starch and 70-80 g/L of total sugar. Under the optimal conditions, a final α-amylase inhibitors concentration of 4726 mg/L was achieved after 168 h.