In this study, chondroitin sulfate was extracted from Oreochromis niloticus bones (OCS) and isolated to three fractions (OCS-1, OCS-2, and OCS-3). The physicochemical properties and structure characterization including monosaccharide, disaccharide compositions, molecular weight (Mw) of OCS were determined by HPAEC, HPLC-SAX, HPGPC, FT-IR spectra, and 1D/2D NMR. Moreover, their thermal properties, crystalline structure, and microstructure were also analyzed. Results showed that their Mw were between 10 kDa and 50 kDa. CS-6 was the predominant disaccharide unit in four OCS, and the CS-4/CS-6 ratios were close to CS from shark cartilage. Besides, the results of antioxidant activity showed that different fractions of OCS had a distinct DPPH radical, hydroxyl radical, and ABTS+ radical scavenging activity. OCS-1 has the highest scavenging activities in DPPH and hydroxyl radical compared with other fractions, which showed a higher medicinal value. Those findings may lay some theoretical basis for the potential application development of OCS.