Abstract: Gardenia Yellow is the prime developed product of the traditional Chinese medicine Gardenia,are widely used in food,pharmaceutical and other fields. In this paper,an optimum system was established to study the alpha-glucosidase inhibitory activity through the single factor on substrate concentration,the amount of enzymes and reaction time. On that basis,the effect of Gardenia Yellow on alpha-glucosidase inhibitory and kinetics of enzyme inhibition were studied. The results showed that the best reaction system of alpha-glucosidase inhibitory activity was as follows:substrate concentration was 3 mmol/L,amount of enzyme was 50 μL and reaction time was 50 min,inhibition level was more significant for the alpha-glucosidase systems originate from pig pancreas and intestinerice,and Saccharomyces cerevisiae,but not for the Bacillus subtilis. Inhibitory characters of Gardenia Yellow on alpha-glucosidase belongs to a competitive inhibition system. Research on hypoglycemic component from Gardenia Yellow and studying the mechanisms had significant practical value and also could broad the application prospects,then of course will lay a solid theoretical basis for the future search of traditional Chinese medicine on treating diabetes.