The extraction methods of flavonoids from bitter Gourd was studied and the biological activity of the extracted material was analyzed in this paper. Bitter Gourd powder was obtained by freezing. Different reagents and methods were studied to extract flavonoids and the biological activity was analyzed. The results show that the best method is using acetone and 75 % of ethanol solution as extraction agent, liquid extraction combined with ultrasonic shock extraction at 60 °C. The results of qualitative analysis show that the extract contains flavonoids. The results of biological activity analysis showed that the crude extracts of acetone and 75 % of ethanol and the purified products of polyamide had different degree of inhibitory effect on Escherichia coli、Staphylococcus aureus、Pseudomonas aeruginosa, and the sensitivity to the three bacteria was: Escherichia coli<Staphylococcus aureus<Pseudomonas aeruginosa.