A suitable extraction method was selected to extract the antibacterial substance from bitter melon and the bioactivity was analyzed in this text. The effective method is extracting with 70 % ethanol, breaking with ultrasonic, extracting with n-butyl alcohol, and then using different concentrations of ethanol for elution from polyamide column. The results of the experiments indicated that the crude extract, the n-butyl alcohol extraction phase and the elution showed obviously antibacterial activity to Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The extracts in this text of bitter melon obviously cleared the H₂O₂. Qualitative analysis showed that the extracts contained saponins, flavonoids, polyphenols and organic acids.