Abstract: HSJ296, a Scenedesmus strain isolated and purified by this lab, can grow heterotrophically in the dark and contains a relatively high amount of α-linolenic acid. Comparisons of heterotrophic growth at different temperatures and concentrations of glucose, or with different nitrogen sources, indicated that the optimal culture conditions included 30℃, 4 g/L urea and 20—40 g/L glucose. As shown with fatty acid composition analyses under different conditions, the fatty acids of HSJ296 were mainly composed of hexadecanoic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2) and α-linolenic acid (α-C18:3), and the α-linolenic acid content in total lipids was between 35%—45% in independent experiments. The fermentation product of Scenedesmus HSJ296 may be used as a fish dietary supplement to supply α-linolenic acid and other nutrients.