In this work, the use of enzymes as alternative catalysts for the production of biodiesel was evaluated. Due to the high price of enzymes, process optimization and economical evaluation is essential to evaluate the feasibility of their use. Two alternatives were proposed. The first one evaluated the use of liquid enzyme while the second alternative analyzed the use of immobilized enzyme for the transesterification of castor oil. Both alternatives were investigated as an integral part of the whole production plant and optimized considering the profit of the process as objective function. For an annual biodiesel production capacity of 250,000 tons, the process with liquid enzymes resulted in higher total investment costs due to the complex steps for the biodiesel separation. However, the higher cost of immobilized enzymes requires that this enzyme is reused at least 300 times to obtain a profit similar to the liquid enzyme-based process, which resulted in a production cost of 0.78 US$/kg of biodiesel and a plant profit of 51.6 million US$/year for the defined plant capacity.