The study focuses on developing a novel perilla seed oil (PeO)-based polyunsaturated fatty acid-rich margarine fat analogue using enzymatic interesterification. PeO is a rich source of omega-3 fatty acids, however, has limited application due to susceptibility to oxidative and thermal degradation. Moreover, low consumption of omega-3 fatty acids in modern diets serves as a major cause for increased prevalence of cardiovascular diseases. The stability of such oils can be improved by techniques like blending and interesterification. However, blending lacks uniformity, which can be overcome by interesterification. The process conditions, namely, enzyme load, temperature, substrate molar ratio, and reaction time, were systematically optimized using response surface methodology. The optimized interesterified fat was characterized for fatty acid and triacylglyceride (TAG) composition. The physicochemical and functional properties, along with oxidative stability, were also evaluated to determine its overall quality. The structured lipid with zero trans and low saturated fat was developed with 50:50 substrate molar ratio of PeO and palm stearin at 54°C within 5.4h at 6.2% w/w concentration of TLIM enzyme. It exhibited 23.8% degree of interesterification, 435.5g hardness, and 33°C slip melting point. The TAG profile of the resulting fat was significantly modified with increased triunsaturated TAGs like LnLnLn and LnLnL but reduced trisaturated TAGs like PPP. The interesterification process lowered tocopherol composition by 3%. The acid, peroxide, IP (Induction Period), and p-anisidine values of PeO significantly enhanced. The thermal behavior of the developed fat was also modified. The textural properties of margarine developed from optimized fat were comparable to commercial margarine, revealing the application of PeO in healthy margarine formulation.