In order to promote the quality of chondroitin sulfate, Sevag reagent was used to deprotein the extraction of chondroitin sulfate through the single factor and the orthogonal tests to optimize the process of deproteinization. The optimal conditions were ratio of chloroform to isoamyl alcohol 21 (V/V), Sevag reagent dosage 1/2 fold compared with the volume of chondroitin sulfate extraction, treated for 25 min and repeated treatment for 3 times. The factors influenced the extraction in the order of ratio of chloroform to isoamyl alchohol,treatment time , treatment times, and Sevag reagent dosage. Under the optimal conditions, the retention rate of chondroitin sulfate was 87.98%, and the deproteinization rate was 52.15%. No specific absorption of 280 nm was observed with ultraviolet (UV) scanning from 190 to 350 nm, and the configuration of chondroitin sulfate was confirmed untransformed with Fourier-transform infrared spectroscopy (FTIR) analysis. The results suggested that this deproteinization method was stable, reliable and suitable for deproteinization of chondroitin sulfate in industry.