Biodiesel fuel was produced by the transesterification of microalgae oil using sodium hydroxide as a catalyst. The oil was extracted from heterotrophic cultivated algae biomass. The transesterification process was optimized using response surface methodologyto increase the yield of methyl esters. The Box-Behnken design and fractional factorial design 2(4-1) points were used to investigate the interaction of process variables, such as the methanol/oil molar ratio, the percentage of sodium hydroxide, the temperature, and the reaction time in the production of biodiesel fuel, and to predict the optimum process conditions for the FAME yield. Based on the results, the optimal conditions for the synthesis of biodiesel fuel were as follows: methanol/oil molar ratio, 7:1; catalyst concentration, 1.0% (by weight of algae oil); temperature, 67 degrees C, and reaction time, 51 minutes. The yield of FAME was confirmed by gas chromatography analysis.