Abstract: In order to optimize the extraction process of moutan cortex anti-vibrio substances and analyze its anti-vibrio components, in the experiment, first, the single-factor test was used to determine the optimal extraction solvent, solvent concentration, extraction temperature, extraction time and liquid-material ratio of moutan cortex anti-vibrio substances. Then, on the basis of the single-factor test, the three influencing factors of the response surface optimization method were selected, and the diameter of the inhibition circle was used as the response value, and the extraction process of anti-vibrio substances of moutan cortex was optimized by the three-factor three-level response surface method; and the optimal extraction conditions were obtained and verified. Finally, by activity tracking method, silica gel column chromatography and high performance liquid chromatography（HPLC） were used to preliminarily analyze the anti-vibrio active ingredients in moutan cortex. The results showed that the best extraction solvent was ethanol solution; the ethanol concentration was 50%; the extraction temperature was 50 ℃; the extraction time was 60 min, and the liquid-material ratio was 25∶1（mL/g）. The extraction temperature, extraction time and liquid-material ratio were selected as the optimization factors of the response surface method, and the extraction conditions were determined as extraction temperature of 50 ℃, extraction time of 52 min, and liquid-material ratio of 24∶1（mL/g）. Under these conditions, the diameter of the inhibition circle was 23.11 mm, which was close to the predicted value of 23.19 mm. Two anti-vibrio active ingredients were identified from moutan cortex active substances, namely paeonol and 1,2,3,4,6-o-pentagallyl glucose. The results suggested that this study successfully optimized the optimal extraction process conditions of anti-vibrio substances of moutan cortex, and determined that the main components of the active substances were paeonol and 1,2,3,4,6-o-pentagallyl glucose.