Background: During fish processing operations, significant fish waste is generated, causing environmental problems. This study compared enzymatic hydrolysis and chemical methods for oil extraction from rainbow trout (Oncorhynchus mykiss) waste and its influence on omega 3 fatty acid profile.Methods: Oil extraction efficiency and fatty acids profile from trout fish head were analyzed by comparing enzymatic method (protease from Bacillus subtilis) and chemical extraction with hexane (soxhlet method) to produce a valuable product by fish waste. The enzymatic hydrolysis and chemical methods for oil extraction from rainbow trout (Oncorhynchus mykiss) waste and its influence on omega 3 fatty acid profile were compared. The contents of fatty acids were analyzed by gas chromatography.Results: No significant difference was noticed between the enzyme treatments with water (200, 100, and 50 mL) and without additional water regarding oil extraction efficiency. The samples without water were economically more affordable because of their lower volume and reducing energy consumption. The oil extraction efficiency with optimum enzymatic method (150 ppm of concentrated protease, without using water) was significantly lower than soxhlet method. Omega-3
content in the optimum biological method (9%) was significantly higher than that in soxhlet method (5.53%). Fatty acids
with high contents of trout head oil in both methods were linoleic acid (18:2) and oleicacid (18:1).Conclusion: Due to the suitable oil extraction efficiency and higher omega-3 fatty acid content of the enzymatic method compared to chemical solvent (hexane) extraction, enzymatic method was preferred as a safe and environment-friendly extraction technique.