In this study, chondroitin sulfates were prepared from tilapia processing by-products (head, backbone, fin and tail) by two-step enzymatic hydrolysis followed by precipitation with cetane pyridine chloride. Their physicochemical properties and structural characteristics were analyzed by ultraviolet (UV) spectroscopy, cellulose acetate electrophoresis, infrared spectroscopy and high performance liquid chromatography (HPLC). The results showed that fish heads and tails mainly contained type C chondroitin sulfate (Δdi6S) at levels of 46.10% and 41.01%, respectively; fish backbones and fins mainly contained type A chondroitin sulfate (Δdi4S) at levels of 71.86% and 69.59%, respectively. The decreasing order of the purity of chondroitin sulfates from these body parts was heads (90.37%), tails (83.33%), backbones (59.76%), and fins (52.01%). Monosaccharide composition analysis showed that chondroitin sulfates from fish heads, backbones, fins and tails were mainly composed of glucuronic acid, glucose and galactosamine in different proportions, and their number-average molecular masses were 51422, 18402, 19481 and 76371, respectively. The types and composition of chondroitin sulfate extracted from different body parts were significantly different. Chondroitin sulfates extracted from tilapia heads and tails were similar to that from shark cartilage, and thus may have the potential to replace chondroitin sulfate from shark cartilage.