In this paper, cartilages of the Amur sturgeon were used as the material to study the optimized protocol for the alkali extraction technology of chondroitin sulfate based on the process of alkali extraction-enzymatic hydrolysis-alcohol precipitation. By single factors (different alkali concentration, alkali liquor temperature, alkali liquor content) and orthogonal test, the effects on yield of product and hexosamine content were studied. The results showed that the optimum alkali extraction technology was as follows: adding 2 times amount of 6% NaOH, and stirring in water at 40 ℃ for 4 h. And the yield of chondroitin sulfate made by this technology reached 40.45%, the quality met the requirement of WS1-C3-0030-2000.The breast cancer cell line (MCF-7 cell) , gastric cancer cell line (MGc803 cell) and hepatoma cell line (SMMC7721) were treated 72 h with the pure and crude samples of sturgeon chondroitin sulfate, and its IC₅₀ were 7.28 mg/mL、7.68 mg/mL、4.25 mg/mL and 8.86 mg/mL、9.27 mg/mL、7.37 mg/mL respectively, its antitumor activities were similar to the pure and rough samples of shark.