Microreactors are efficient with regard to the continuous production of biodiesel, because of their enhanced mass transfer. In this study, a novel structure of microchannel reactor was studied to synthesize biodiesel from soybean oil via alkali-catalyzed transesterification. Response surface methodology (RSM) was applied to evaluate the relationship between biodiesel yield and reaction parameters, such as residence time, reaction temperature, catalyst amount, and molar ratio of methanol to oil. A three-level four-factor Box-Behnken design (BBD) was used to fit the available response data to a second-order polynomial regression model. Under the optimum conditions of a residence time of 14.9 s, a methanol/oil molar ratio of 8.5, 1.17 wt % KOH, and 59 degrees C, the biodiesel yield reached 99.5%. The effect of moisture and free fatty acid on biodiesel production were also explored.