<div><p>Large-scale purification of omega-3-rich galactolipids is of growing interest but relies on hazardous solvents. This study presents a scalable and low-toxicity approach using ethanol-based extraction from Medicago sativa and Nannochloropsis oceanica. Pigments were removed using activated carbon, and non-lipid compounds were eliminated via ethyl acetate partitioning. Fractionation was performed on silica gel using n-butyl acetate, ethyl acetate, and acetone. Ethyl acetate fraction yielded 0.57 g (93.6 % α-linolenic acid) and 0.91 g (55.9 % eicosapentaenoic acid) of monogalactosyldiacylglycerols per 100 g of dry biomass of M. sativa and N. oceanica, respectively. Acetone fractions were further purified by anion exchange chromatography using ethanol, yielding 0.41 g (79.5 % α-linolenic acid) and 0.75 g (29.6 % eicosapentaenoic acid) of digalactosyldiacylglycerols per 100 g of dry M. sativa and N. oceanica, respectively. The high purity of galactolipid fractions was demonstrated by high-performance liquid chromatography and high-performance thin-layer chromatography. To the best of our knowledge, this is the first complete purification process for galactolipids that relies exclusively on green solvents and is suitable for preparative scale.</p></div>