Fatty acids fractionation has enabled the introduction of many fatty acids into new food/feed applications. Yet, a simple, efficient, and low-cost process to separate biomass oily feedstocks into valuable fatty acids fractions remains a challenge. Herein, a new, efficient, and simple fractionation method is developed to obtain enriched polyunsaturated fatty acids fraction from fish waste oil using acetone for crystallization at low-temperature followed by urea complexation. This method obtained two fractions, one enriched in polyunsaturated fatty acids, and the other enriched in saturated fatty acids. The fraction enriched in polyunsaturated fatty acids contained oleic, eicosapentaenoic, and docosahexaenoic acids with mass percentages of 58, 20, and 22%, respectively. While the fraction enriched in saturated fatty acids contained 78, 12, 5, and 5 wt. % palmitic, myristic, oleic, and stearic acids. The solvent was recovered from the liquid phase with an efficiency of 80% and reutilization of it obtained almost identical results.