The aim of this study is to produce new medium-long-medium structured triacylglycerols (MLM-STAGs) containing caprylic add (CA) at the sn-1,3 positions, and long chain saturated fatty acids (LCSFAs) and bioactive fatty acids at the sn-2 position via two-step enzymatic process by Lipozyme TL-IM, using autotrophic microalgae (Isochrysis galbana) TAGs. In the first step, the ethanolysis of microalgae oil TAGs for 2-monoacylglycerols (2-MAGs) production are performed in the presence of water content of 5 wt% at ethanol/oil mole ratio of 24:1 with Lipozyme TL-IM at 25 degrees C for 12 h. In the larger-scale production of 2-MAGs, the 2-MAGs yield and the purity of the purified MAGs attained are 31.44-36.08 wt% and 91.57 mol%, respectively. In the second reaction, these 2-MAGs are esterified with caprylic acid (CA) in n-hexane system to synthesize almost pure MLM-STAGs with the recycled Lipozyme TL-IM under the optimized conditions. The final MLM-STAGs contain 64.28% CA and 13.35% omega-3 polyunsaturated fatty acids. The CA-LCSFAs-CA (43.01%) and novel CA-stearidonic add (SDA, C18:4 omega-3)-CA (23.88%) are the major MLM-STAGs present. In all, Isochrysis galbana-derived oil containing SDA is a promising source for producing high-value microalgal bioproducts by two-step enzymatic process in a cost-effective manner.