Microbial oil, as raw material for biodiesel, can be produced by Trichosporon cutaneum B3 using cassava starch hydrolysate. Batch cultures demonstrated that there was little inhibitory effect with the concentration of cassava starch hydrolysate up to 90 g/L. The favorable initial pH, C/N molar ratio, nitrogen source and its concentration were 6.0, 116, yeast extract and 3.0 g/L, respectively. Under the optimized conditions, dry biomass reached 15.2 g/L and lipid content reached 40.9% after culture for 144 h in flask. Batch cultures in a 2 L stirred-tank fermenter were run for 44 h and resulted in dry biomass, lipid content and lipid yield of 28.7 g/L, 42.8% and 12.27 g/L, respectively. The chemical compositions of biodiesel prepared from lipids of T. cutaneum B3 mainly included palmitic acid methyl ester, stearic acid methyl ester, oleic acid methyl ester and linoleic acid methyl ester etc., and its main physicochemical properties were in compliance with relevant national diesel standards. Therefore, the biodiesel prepared from lipids of T. cutaneum B3 can serve as a potential fossil fuel alternatives.