The half inhibitory concentration (IC₅₀) of alpha glucosidase inhibitors was detected based on a microplate assay method in this study in order to find the effective inhibitors for preparing hypoglycemic functional food. The reaction conditions were optimized by the four aspects, i.e., the enzyme concentration, the detection wavelength, the substrate concentration and the reaction time. The results showed that the optimum enzyme concentration ranged from 2.0 mg/mL to 3.0 mg/mL, and waves 520 nm and 492 nm were optimum among the five waves, 405, 450, 492, 520, 630 nm. Moreover, the suitable substrate concentration of sucrose ranged from 20 mmol/L to 40 mmol/L, and the appropriate reaction time was about 20~30 min.