Abstract: This article mainly adopted response surface methodology to optimize the extracting technology of chondroitin sulfate by enzyme. Chondroitin sulfate with high purity was obtained after trichloacetic acid for deproteinization, alcohol precipitation and column chromatography for further isolation and purification. High performance gel filtration chromatography was used to identify purity of chondroitin sulfate. Infrared spectrum and nuclear magnetic resonance were used to analyze its isomerism. The results show that the optimal condition of extraction was 55.68℃ of temperature, 6.40 of pH and 3.85 h of extraction time. The extraction rate of glucuronic acid and chondroitin sulfate is 9.82% and 28.05% respectively. The data of high performance gel filtration chromatography exhibited that obtained chondroitin sulfate is of highly purity (99.01%). Infrared spectra and nuclear magnetic resonance exhibited that it is chondroitin-4-sulfate.