Objective: To optimize the refining process of crude fish oil from mandarin fish viscera and to improve the quality of fish oil. Methods: With phosphoric acid volume fraction, addition amount of NaOH and activated clay as influencing factors, the acid value and peroxide value as the evaluation indexes, the refining process was optimized by Box-Behnken response surface analysis. The sensory quality, physicochemical indexes and fatty acid composition of fish oil before and after refining process were analyzed. Results: The optimal refining conditions were obtained as follows, phosphoric acid volume fraction of 50%, addition amount of 2% NaOH, addition amount of 15% activated clay. Under the optimal refining condition, the acid value and peroxide value of refined fish oil were (0.94±0.03) mg/g and (0.12±0.01) g/100 g in accordance with the predicted values, which indicates regression equation is significant. The sensory quality, physicochemical indexes and physical properties of fish oil after refining process were improved effectively, reaching the second-grade or above standards of refined fish oil of China’s aquatic industry (SC/T 3502—2016). The fatty acid composition didn’t change significantly during the refining. The refined fish oil was dominated by unsaturated fatty acids, mainly 11-Octadecenoic acid (36.73%) and 9,11-Octadecadienoic acid (27.02%). Conclusion: This research reveals the refining process and quality changes of fish oil, and can provide a theoretical basis for the effectively development and utilization of mandarin fish visceral byproducts.