The production of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) concentrate from cobia liver oil by acetone fractionation of fatty acid salts was investigated in this study. A three-level-three-factor Box-Behnken design was used to evaluate the effects of reaction time, amount of NaOH added, and acetone ratio on the responses (DHA and EPA content and recovery). The results showed that the amount of NaOH added was the most important factor in the process. The DHA content showed an inverse relation with EPA content and recovery, whereas its content increased proportionally with the amount of NaOH added. With a reaction time of 1.51 h, amount of NaOH added at 0.65 times the molar equivalent of free fatty acid (FFA), and acetone ratio at 13.92, a maximum recovery of DHA + EPA was 98.14%, and the obtained concentrate contained 71.23% DHA + EPA. Finally, the lipase-catalyzed esterification of the DHA + EPA concentrate with glycerol was carried out. The acetone fractionation of fatty acid salts is an efficient technique for producing DHA +EPA concentrate. The DHA +EPA concentrate can be used as starting materials for the production of functional lipids to provide n-3 polyunsaturated fatty acids to the consumers.