Enzymatic synthesis of biodiesel by the transesterification of canola oil and methanol in high pressure carbon dioxide [HPCO(2): near-critical and supercritical carbon dioxide (NcCO(2) and ScCO(2))] was optimized using response surface methodology (RSM). RSM based on 5-level-5-factor central composite rotatable design (CCRD) was used to evaluate the effects of temperature, pressure, enzyme loading, substrate molar ratio, and time on the conversion to biodiesel by transesterification. Finally, batch reactions for biodiesel synthesis were preformed in a 100 mL and 7 L high-pressure stirred batch reactors.