Alkali-catalyzed and supercritical methanol transesterification were used to produce biodiesel from Chinese cottonseed. Fourier transform infrared and gas chromatography/mass spectrometry were used to identify the compositions of cottonseed oil biodiesel samples. Six major compositions of the biodiesel samples were identified by the retention time and the fragmentation pattern data of GC/MS analysis. The yields of biodiesel samples obtained from alkali-catalyzed reaction and supercritical methanol transesterification were 98.4 and 99%, respectively. In comparison to the alkaline transesterification, the supercritical methanol transesterification combining with two-phase extraction was more suitable for biodiesel production due to shorter reaction time, less purification steps, and lower cost.