Summary This study evaluated the quality of oil extracted from yellowfin tuna (Thunnus albacares) by enzymatic hydrolysis (EHO) compared with oil extracted by traditional methods, such as the physical method of cooking and pressing after fishmeal production and the chemical solvent method. The oil extracted by EHO presented the lowest acidity (1.96% oleic acid) and peroxide indexes (5.14 mEq O2 kg−1 of oil) and the highest levels of eicosapentaenoic acid (6.05g 100 g−1) and docosahexaenoic acid (27.15 g 100 g−1), two omega‐3 fatty acids with high nutritional value. Importantly, oil extraction from yellowfin tuna heads using EHO produced oil rich in omega‐3s with no oxidation. This study shows that this extraction method greatly increases the value of fish by‐products and increases the competitiveness of the fishing industry. Enzymatic extraction of oil from yellowfin tuna (Thunnus albacares) by‐products: a comparison with other extraction methods.