Structured lipids (SLs) containing DHA, EPA, and DHA+EPA were synthesized via enzymatic acidolysis using VCO as the substrate in n-hexane. Commercially available enzymes Lipozyme TL IM (produced from Thermomyces lanuginosus, a 1,3-specific lipase), Lipozyme IM60 (produced from Rhizomucor miehei, a 1,3-specific lipase), and non-specific lipase from Candida rugosa (powder) were used as biocatalysts. The T. lanuginosus lipase was chosen to evaluate the effects of various parameters on the incorporation of PUFAs into VCO and to optimize the process. As the enzyme load increased from 1 to 4%, the incorporation of omega-3 PUFAs also increased; however, it decreased when the enzyme load was further increased to 6%. The incorporation of these fatty acids increased with reaction time from 12 to 36h but decreased at 48h. Similarly, the incorporation increased with temperature from 35 to 45 ^oC, but decreased at 55 and 65 ^oC. The highest incorporation rates of DHA (18.91%), EPA (30.38%), and DHA+EPA (34.64%) were achieved at a mole ratio of 1:3 (VCO to DHA or EPA) or 1:3:3 (VCO to DHA+EPA), with a 4% enzyme load, 36h incubation time, and a temperature of 45 ^oC. A central composite design (CCD) with three levels and three factors—reaction temperature (35, 45, 55 ^oC), enzyme amount (2, 4, 6%), and reaction time (24, 36, 48h)—was used to model and optimize the reaction conditions via response surface methodology (RSM). Under optimal conditions of 3.3% T. lanuginosus enzyme, 42.22°C, and 33.38h, the incorporation rates were 32.92% for DHA, 44.48% for EPA, and 47.04% for DHA+EPA in VCO.