In the present study, response surface methodology (RSM) involving central composite design (CCD) was applied to optimize the reaction parameters of biodiesel production from yellow mustard (Sinapis alba L.) seed oil during the single-step transesterification process. A total of 30 experiments were designed and performed to determine under the effects of variables on the biodiesel yield such as methanol to oil molar ratio (2:1–10:1), catalyst concentration (0.2–1.0 wt.% NaOH), reaction temperature (50–70°C), and reaction time (30–90 min). The second order polynomial model was used to predict the biodiesel yield and coefficient of determination (R2) was found to be at 0.9818. The optimum biodiesel yield was calculated as 96.695% from the model with the following reaction conditions: 7.41:1 of methanol to oil molar ratio, 0.63 wt. % NaOH of catalyst concentration, 61.84°C of reaction temperature, and 62.12 min of reaction time. It is seen that the regression model results were in agreement with the experimental data. The results showed that RSM is a suitable statistical technique for optimizing the reaction parameters in the transesterification process in order to maximize the biodiesel yield.