Chinese sturgeon was an endangered cartilaginous fish. The success of artificial breeding has promoted it to a food fish and it is now beginning to provide a new source of cartilage for the extraction of chondroitin sulfate (CS). However, the structural characteristics of sturgeon CS from different tissues remain to be determined in more detail. In this study, CSs from the head, backbone, and fin cartilage of Chinese sturgeon were individually purified and characterized for the first time. The molecular weights, disaccharide compositions, and oligosaccharide sulfation patterns of these CSs are significantly different. Fin CS (SFCS), rich in GlcUAα1-3GalNAc(4S), has the biggest molecular weight (26.5 kDa). In contrast, head CS (SHCS) has a molecular weight of 21.0 kDa and is rich in GlcUAα1-3GalNAc(6S). Most features of backbone CS (SBCS) are between the former two. Other glycosaminoglycan impurities in these three sturgeon-derived CSs were lower than those in other common commercial CSs. All three CSs have no effect on the activity of thrombin or Factor Xa in the presence of antithrombin III. Hence, Chinese sturgeon cartilage is a potential source for the preparation of CSs with different features for food and pharmaceutical applications.