Biodiesel is increasingly being considered as an alternative to the fossil fuel as it is renewable, nontoxic, biodegradable, and feasible for mass production. Biodiesel can be produced from various types of vegetable oils. Calophyllum inophyllum seed oil (CSO) is among the prospective nonedible vegetable oils considered as a raw material for biodiesel synthesis. The most common process of the biodiesel manufacturing is the transesterification of vegetable oils which results in glycerol as a by-product. Thus, product purification is necessary. In this work, an alternative route to biodiesel synthesis through interesterification reaction of vegetable oil and ethyl acetate was conducted. By replacing alcohol with ethyl acetate, triacetin was produced as a side product rather than glycerol. Triacetin can be used as a fuel additive to increase the octane number of the fuel. Therefore, triacetin separation from biodiesel products is needless. The interesterification reaction is catalyzed by an alkaline catalyst or by a lipase enzyme. In this study, biodiesel synthesis was carried out using a lipase enzyme since it is a green and sustainable catalyst. The interesterification reaction of CSO with ethyl acetate in the presence of a lipase catalyst was conducted using the molar ratio of CSO and ethyl acetate of 1:3. The reaction time, lipase catalyst concentration, and reaction temperature were varied at 1, 2, 3, 4, 5 h, 10%,15%, 20%, and 30 °C, 40 °C, 50 °C, 60 °C, respectively. The experimental results were also analyzed using response surface methodology (RSM) with the Box–Behnken design (BBD) model on Design Expert software. Data processing using RSM revealed that the highest conversion within the studied parameter range was 41.46%, obtained at a temperature reaction of 44.43 °C, a reaction time of 5 h, and a lipase catalyst concentration of 20%.