The alga Chlorella protothecoides is known to produce oil suitable for biodiesel preparation by heterotrophic cultivation in media containing glucose as a carbon source. In this study, sugar cane juice was used as alternative carbon supply for oil production. As a result, the highest oil content of 53.0% by cell dry weight was achieved. Fermentation in a 5 L bioreactor showed that algae using sugar cane juice hydrolysate (SCH) grow faster thin) that using glucose. Conversion ratios of sugar/biomass and sugar/oil using SCH were 15.2 and 8.8% higher than that using glucose, respectively. Biodiesel prepared from algal oil by transesterification is mainly composed of 9-octadecenoic acid methyl ester, 9,12-octadecadienoic acid methyl ester, and hexadecenoic acid methyl ester. Our results suggest that sugar cane is a good feedstock for biodiesel production. Response surface methodology upon exploring the effect of C/N and concentration of yeast extraction (YE) on the yield of biomass and oil was performed. The optimal production with the highest output-cost coefficient of 0.061 +/- 0.004 was achieved when C/N was 26.9 and YE was 0.60 g L-1.