In this study, we investigated enzymatic pre-treatment of grease trap waste (GTW) as an environmentally beneficial procedure for biodiesel production. Different enzymes, both commercial and newly designed industrial enzymes, were used to reduce the free fatty acids (FFA) level of GTW through an esterification reaction. The process conditions were optimized using response surface methodology with central composite design parameters. A set of 30 experiments, for both batch and continuous flow reactors, were designed to identify the optimal process conditions in which the highest conversion of FFA is achieved. Within the range of the selected operating conditions, the optimized values of reaction temperature, catalyst quantities, ethanol to oil molar ratio, and reaction time for the batch reactor, in which FFA level was reduced to 31.5 %, were found to be 70 ◦C, 4.5 wt%, 3:1, and 25 min respectively. A significant improvement in the reduction of FFA, of which FFA amount is only 9.9 %, was obtained in the flow reactor when using the commercial enzyme (T = 57 ◦C, catalyst loading 4.85 %, ethanol to oil ratio 2:1, t = 25 min). In addition to achieving higher conversion, the continuous-flow experiments saved time since the entire series of experiments were completed in