The structure of isoflavone moiety in soybean could be altered by thermal treatment. Thus, this study analyzed the isoflavone profiles of raw soybean extract (RSe, no pretreatment), blanched soybean extract (BSe, at 85oC for 3 min), and cooked soybean extract (CSe, at 95oC for 30 min) and measured their α-glucosidase inhibitory activities. The content of malonylglucosides in RSe decreased considerably during their transformation into BSe and CSe. The total content of isoflavones in RSe and BSe was fairly similar, although there was a significant difference in the respective values for RSe and CSe. It was concluded that the cooking treatment significantly impacted soybean isoflavone content and, eventually, one-quarter of isoflavones were lost in CSe. According to authentic isoflavone tests, the inhibition of α-glucosidase by isoflavone aglycones was more effective than that of the corresponding isoflavone glucosides. The α-glucosidase inhibitions were observed in the order of BSe (70.0%), CSe (53.3%), and RSe (32.3%). However, it was challenging to identify which isoflavone derivative had contributed to the phenomenon mentioned above. Hence, the blanching of soybean seemed to be more appropriate than cooking for preparing soybean extract to inhibit α-glucosidase due to the higher loss of isoflavone during cooking.