Omega-3 is a polyunsaturated fatty acid (PUFA), which is an essential nutrient for health. Through this, the x3-PUFA concentration has increasingly gained interest. In this study, the concentration of x3-PUFA of Lantern fish oil was investigated through utilisation of membrane separation. Between tested membranes, it was observed that ETNA01PP separates x3-PUFA more selectively. Additionally, effects of main parameters affecting the x3-PUFA concentration, including temperature (20-40°C), pressure (3-5 bar) and stirring rate (0-200 rpm), were evaluated through Box-Behnken design of experiment. According to ANOVA, all these parameters showed significant impact on x3-PUFA concentration. To determine the optimal process parameters, the Derringer's desirability prediction tool was employed in which the highest x3-PUFA concentration of 35.1058 wt. % was estimated. The obtained optimal condition was found to be the temperature of 36.19°C, pressure of 4.82 bar and stirring rate of 43.01 rpm with a desirability value of 0.99.