Abstract: Objective: To enhance the comprehensive utilization value of bovine bone by-products, improve the yield of chondroitin sulfate (CS), and clarify the cartilage damage protection activity of CS from different sources, this study investigated the preparation process and bioactivity of CS derived from bovine bone by-products. Methods: Using the CS yield as the evaluation index, the alkaline extraction process of nasal bone-derived CS was optimized through response surface methodology (RSM) based on single-factor experiments. An ultrafiltration-spray drying technology was employed to prepare nasal bone-derived CS (BCS-1) from the extract. Structural differences among BCS-1, bovine scapular bone-derived CS (BCS-2), and bovine tracheal cartilage-derived CS (BCS-3) were analyzed. A zebrafish cartilage injury model induced by dexamethasone was used to compare the differences in cartilage damage protection activity among CS from different sources. Results: The optimal alkaline extraction conditions for nasal bone-derived CS were: solid-to-liquid ratio 1:15.5, temperature 27.2 ℃, and extraction time 15.7 hours, under which the CS yield reached 22.52%±0.17%. The prepared BCS-1 met the moisture content, purity, and specific optical rotation standards of the Chinese Pharmacopoeia. Infrared spectroscopy showed that the structure of CS from different bovine sources was consistent with the sodium CSA reference standard (CP). Disaccharide composition analysis revealed significant differences among CS from different sources, with the AC ratio (the ratio of chondroitin sulfate A to chondroitin sulfate C) of BCS-1 being close to that of CP. At concentrations of 500, 1000 and 2000 μg/mL, the cartilage regeneration rates of BCS-1 were 61.59%±19.92%, 66.98%±25.78%, and 71.75%±8.57%, respectively, compared to 47.02%±12.13%, 52.56%±14.06%, and 53.40%±15.47% for BCS-2 and 43.87%±13.52%, 45.86%±13.52%, and 57.61%±17.06% for BCS-3. Conclusion: The alkaline extraction process for CS from bovine nasal cartilage established by this study achieves a high yield. The extracted solution can be used to produce Chinese Pharmacopoeia-grade CS via ultrafiltration-spray drying technology, which is safer compared to the organic solvent precipitation method. BCS-1, BCS-2, and BCS-3, derived from different bovine bone by-products, all exhibit excellent protective activity against cartilage damage. Among them, BCS-1 demonstrates the best protective activity, indicating significant development and application potential.