Biodiesel has become increasingly attractive because of its environmental benefits and production from renewable resources. The use of Jatropha curcas L. oil as the feedstock for biodiesel production has attracted growing interest because it is a non-edible oil. Herein, easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was used to chemically characterize, at the molecular level, J. curcas L. oil and its biodiesel via monitoring its triacylglycerols (TAG), free fatty acids (FFA), and profiles of fatty acid methyl esters (FAME). The results provide further support for those obtained by the standard parameters of fuel quality. The EAST MS analysis described herein is simple, requires only a tiny droplet of the sample, and is conducted without any pre-separation or chemical manipulation steps.