Oil from Jatropha seeds was extracted by supercritical CO2. A catalytic transesterification reactor was employed for biodiesel production from extracted oil in which fatty acids like palmitic, stearic, oleic, and linoleic acid were converted to fatty acid methyl esters (FAMEs) with sodium methoxide as the catalyst. Gas chromatography-flame ionization detector (GC-FID) analysis identified and quantitatively determined the amount of FAMEs. Response surface methodology (RSM) was applied to find the optimal operating conditions in order to maximize the biodiesel yield. Under the RSM-predicted optimum conditions, the maximum yields of four individual FAMEs and their combination as biodiesel were determined. The RSM model demonstrated that the linear and square terms of four variables and the interaction of flow rate and dynamic time significantly influence the biodiesel yield.