Fish oil that meets quality standards is obtained by degumming, deacidifying, bleaching, and deodorization processes in conventional fish oil production. Less attention has been paid to unhealthy ingredients such as cholesterol, cholic acid, and trans fatty acids. In this study, the traditional fish oil production process was simulated, and eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), cholesterol, total cholic acid, and trans-fatty acid (C18:1tran, C18:2trans, C18:3trans）content were studied, while the quality of fish oil product was ensured. The results showed that the acid value, peroxide value, and iodine value of refined fish oil was 0.53 mg KOH/g, 4.35 mmol/kg and 139.60 g/100 g, respectively, which reached the primary standard of the polyunsaturated fish oil industry (SC/T 3502-2000). The total relative EPA and DHA content were not significantly changed during the refining process, remaining around 26%, indicating that refining has little effect on EPA and DHA content. Cholesterol and total cholic acid content exhibited a downward trend over the refining process; their final content in refined fish oil were 240 mg/100 g and 3 mg/100 g, respectively, and their removal rates were 57.4% and 97.2%, respectively. Trans fatty acid content increased during the refining process, and reached 1.42% in the final oil product. The above results indicate that the conventional refining process still has some limitations, and should be improved to further enhance the quality of fish oil.