Abstract Human milk fat analogues containing stearidonic acid (SDA) were produced by enzymatic acidolysis reactions between tripalmitin and free fatty acids (FFA) obtained from hazelnut oil and commercial oil mixture containing Echium oil. Lipozyme® TL IM, immobilized sn-1,3 specific lipase was used in the enzymatic reactions. Response surface methodology (RSM) was applied by using central composite circumscribed design with five levels and three factors. The factors chosen were: Reaction temperature [55–65 °C], reaction time [4–12 h] and substrate molar ratio [3–5 mol/mol (total FFA/tripalmitin)]. Good quadratic models were obtained for the incorporation of SDA and oleic acid. Optimal conditions generated from the models were determined as 60 °C; 8 h, 4 mol/mol for temperature, time and substrate molar ratio, respectively. The models were verified at optimum conditions and furthermore scale-up synthesis of structured lipids (SLs) was performed. Stearidonic and oleic acid contents of the SL from scale-up production were found to be 2.0 and 22.9 mol/100 mol total FA respectively, with 46.2 mol/100 mol total FA of palmitic acid content located at sn-2 position. Besides, oxidative stability and melting profile of final SL were determined for characterization. SL had 6.1 h of induction time, and also had a wider melting range compared with tripalmitin.