The essential fatty acid a-linolenic acid is vital for human physiology and health. The green alga Scenedesmus sp. HSJ296 can accumulate large amounts of alpha-linolenic acid. In this work, we optimized the fermentation of Scenedesmus sp. HSJ296 in a 50-L fermenter, resulting in a biomass productivity of 3.12 +/- 0.45 g L (-1) d (-1). We then extracted lipids from the biomass by supercritical fluid CO2 extraction with 95% ethanol as entrainer, achieving a lipid extraction rate as high as 73.47 +/- 4.28%. After decolorization and enrichment by lipase hydrolysis, and low-temperature solvent crystallization, the content of unsaturated fatty acids in the lipid fraction from the biomass was enriched up to 92.5 +/- 1.2%, with alpha-linolenic acid accounting for 62.6 +/- 0.9%. The saturated fatty acids mainly consist of palmitic acid (C16:0, 3.1 +/- 0.1%) and stearic acid (C18:0, 0.9 +/- 0.1%). The remaining saturated fatty acid fraction, with relatively higher saturated fatty acid content, can be used as raw material for biodiesel production. Overall, we established an optimized alpha-linolenic acid production process that provides an important theoretical and technical basis for microalgae-based production of high-value lipids.