Cassia fistula seed oil (CFSO) was investigated for the first time as an non-conventional feedstock for the production of biodiesel. The yield of CFSO is 27.03 +/- 2.3% (weight in seed basis) and has an low acid value of 0.92 (mg KOH/g). The fatty acid composition of CFSO predominantly contains stearic acid, oleic acid, myristic acid, and fumaric acid. Biodiesel was prepared by sodium hydroxide-alkaline catalyzed transesterification of oil with methanol, and the reaction conditions were optimized. The optimal biodiesel yield of (91.34 +/- 2.05%) was obtained. The NMR spectra of the methyl esters of C. fistula seed oil were reported.