Biodiesel, an alternative to traditional diesel, is essential for the sustainability of long-term energy supplies and often synthesized through a non-alcoholic route called interesterification. The described synthesis method facilitates the modification of oil and fat by exchanging acyl radical groups between triglyceride and alcoholic acid (alcoholysis), fat (acidolysis), or ester (transesterification). Therefore, this research aimed to determine the effect of the reactant ratio between crude palm oil (CPO) and dimethyl carbonate (DMC), along with the use of an eco-enzyme catalyst, on biodiesel characteristics. The CPO:DMC ratio was 1:1.5, 1:2, 1:2.5, and 1:3, while the immobilized eco-enzyme catalyst was 2%, 3%, 4%, 5%, and 6% of CPO mass. The results showed that interesterification with a 1:3 reactant ratio using a 4%wt catalyst was the best procedure, producing biodiesel yield of 73.65%, density of 0.860 g/mL, viscosity of 4.63 mm2/s (cSt), flash point of 113 °C, calorific value of 34.454 MJ/kg, and cetane number of 70.6%.