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Microalgae have attracted growing interest all around the world due to their potential applications in multiple sectors of industry, such as energetics, nutraceuticals, pharmaceuticals, agriculture, and ecology. Concepts of biorefinery of microalgae lipids for biodiesel production coupled with other applications have been suggested in several studies. However, very few studies focus on overcoming the degree of unsaturation of microalgae lipids using methods of fractionation. This study presents a method for obtaining two oil fractions from microalgae Chlorella sorokiniana suitable for food and biofuels via urea complex formation with further production of a long-chain PUFA concentrated oil suitable for the nutraceutical industry. A DHA–EPA-rich fraction was obtained from the dry microalga biomass using a succession of extraction, urea-complexation, fractionation, and esterification with glycerol. Analytical and organoleptic methods were used to assess the quality of the final product. Results show that the urea-complexation method allowed the obtaining of two lipid fractions with different fatty acid profiles. The urea complexed fraction (UCF) contained a majority of saturated fatty acids (54.46%); thus, it could find applications in the biofuels or food industry. The non-urea complexed fraction (NUCF) was rich in polyunsaturated fatty acids (PUFA) (81.00%), especially long-chain PUFA with 16.52% EPA and 35.08% DHA. The recovery rates of EPA and DHA in the NUCF reached 59% and 87.14%, respectively. Finally, the physicochemical and organoleptic characteristics of the DHA–EPA oil concentrate were determined and found conform to the norms recommended by the WHO/FAO standards for edible oils and the Russian State Standard GOST 1129-2013.

Obtaining DHA–EPA Oil Concentrates from the Biomass of Microalga Chlorella sorokiniana