Patin (Pangasius micronemus) fish oil is rich in omega-3 fatty acids such as α-linolenic acid and eicosapentaenoic. To obtain the benefits of omega-3, it is necessary to develop an efficient extraction method for patin oil (PO). This study aimed to develop the microwave-assisted extraction (MAE) method for fish oil from patin. Three independent variables with three levels were priorly evaluated using Box–Behnken design, including temperature (30, 60, 90°C), solvent composition (30, 60, 90% of ethyl acetate in methanol), and solvent to sample ratio (10:1, 15:1, 20:1). The significant variables were then optimized by response surface methodology (RSM). A secondorder quadratic model of RSM suggested an extraction at 60°C with 56% ethyl acetate in methanol as an extraction solvent and a solvent-to-sample ratio of 20:1. A kinetics study under the optimum MAE conditions approved the complete recovery (38.84%) starting at 15 min of extraction time. The high precision of the MAE process was confirmed by the coefficient of variation less than 3%. Additionally, the microwave-produced fish oil was characterized by gas chromatography-mass spectrometry (GC-MS) to contain α-linolenic acid and eicosapentaenoic as omega-3. Henceforth, it has been demonstrated that microwave-assisted fish oil extraction developed in this study is efficient for high-quality PO production.