Perilla seed oil (PSO) is known to be rich in alpha-linolenic acid, but higher sensitivity to oxidation limits its application as cooking oil. This study aimed to synthesize structured lipids (SL) from PSO and palm olein (PO) using enzymatic interesterification (EIE) process. The response surface methodology was used to determine the optimal reaction conditions and results in development of SLs rich in omega-3 fatty acids with improved thermal and oxidative stability. The well-fitted model was successfully established and results demonstrated that the optimum conditions for the synthesis of SL were achieved with a 70:30 (PO:PSO) substrate molar ratio, 8% of enzyme concentration, and 6.6 h of reaction time at 35 °C temperature. The SL produced under these conditions had 24.7% of degree of interesterification (DI), 1.5 of MUFA/PUFA ratio, and 0.79 of omega-6/omega-3 fatty acid ratio. The process of EIE lowered the total tocopherol content by 8.81%. However, the results of oxidative parameters such as peroxide value, p-anisidine value, induction time, and TGA indicated that the EIE process exhibited minimal impact on oxidative stability. Furthermore, the absence of peak at 966 cm−1in Fourier transform infrared spectroscopy (FTIR) analysis confirmed that the developed SLs were free from trans-fat. EIE leads to the formation of new triacylglycerol (TAG) species analyzed by the equivalent carbon number method which is further confirmed through change in the melting and crystallization behavior of SL. Therefore, EIE has the potential to produce trans-fat-free SLs rich in omega-3 fatty acids using PSO and PO with desirable physicochemical and nutritional properties.