Enzymatic methanolysis of canola oil in the solvent-free system was studied in a packed-bed reactor (PBR) using small pieces of loofa plus Novozym 435. Response surface methodology (RSM) was applied to determine the effect of the transesterification conditions, namely flow rate of substrate (x₁), temperature (x₂) and methanol to canola oil molar ratio (x₃) as the regressors, on the methyl ester production. A central composite design (CCD) was employed to optimize the reaction. A second-order polynomial multiple regression model was chosen and analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.996, thus adjustment of the model with experimental data was ensured. The methyl ester yield increased as the flow rate of the reaction mixture in the PBR increased from its low to the middle level thereafter, increasing the flow rate corresponded to decreasing the yield. The same trends of changes were observed for the other two factors. The optimum process conditions for biodiesel production in the PBR were found to be: x₁ = 6.3 mL/min, x₂ = 38 °C and x₃ = 4.3. The same batch was successfully used repeatedly in the PBR for six enzymatic cycles (432 h), where the methyl ester yield was maintained above 97%.