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Cobia, Rachycentron canadum, is a medium-size marine fish with emerging global potential for offshore aquaculture. The processing waste, cobia liver, is a raw material rich in polyunsaturated fatty acid oils. In this study, an environmentally friendly green process, aqueous extraction (AE), was used to extract the cobia liver oil. The effect of cooking time and substrate water ratio on the oil extractability was investigated herein. The cooking time of 15 min, and substrate water ratio of 1:2 obtained the highest extraction efficiency. However, the oil extractability was only 18.8%. Thus, enzyme-assisted aqueous extraction (EAAE) was used to increase oil extractability and recovery of protein hydrolysates. The commercial proteases—including alcalase, papain, trypsin, and pepsin—were employed in pretreated cobia liver in order to increase oil release during AE. The EAAE results showed that maximum oil extractability was 38% by papain pretreatment. EAAE greatly improved the extraction efficiency; the oil extractability was double than that of AE (18.8%). The fatty acid profiles revealed that ω-3 polyunsaturated fatty acid contents of extracted oil obtained from AE and EAAE were 21.3% and 19.5%, respectively. Besides, the cobia liver hydrolysates obtained from EAAE by alcalase, papain, pepsin, and trypsin pretreatment showed scavenge DPPH radical activity with EC50 values of 0.92, 1.03, 0.83, and 0.53 mg, respectively. After in vitro simulated gastrointestinal digestion, the protein hydrolysates exhibited scavenge DPPH radical activity with EC50 values of 1.15, 1.55, 0.98, and 0.76 mg for alcalase, papain, pepsin, and trypsin, respectively. The study showed that the EAAE process can be used for extracting fish oil from fish waste while simultaneously obtaining the protein hydrolysates with antioxidant activity. [ABSTRACT FROM AUTHOR]