Heterotrophic fermentation of microalgae has been shown to accumulate high amounts of microalgal lipids, which are regarded as one of the most promising feedstocks for sustainable biodiesel production. To increase the biomass and reduce the cost of microalgal culture, the purpose of this study was to evaluate the possibility of using cassava starch hydrolysate (CSH) instead of glucose as carbon source for heterotrophic culture of Chlorella protothecoides in flasks. First, the two-step enzymatic process of hydrolysis of cassava starch by alpha-amylase and glucoamylase was optimized; the conversion efficiency for cassava starch was up to 97.7%, and over 80% of CSH was glucose. Subsequently, we compared heterotrophic cultures of C. protothecoiedes using glucose or CSH as carbon source. The results demonstrated that when using CSH as the organic carbon source, the highest biomass and the maximum total lipid yield obtained were 15.8 and 4.19 g/L, representing increases of 42.3 and 27.7%, respectively, compared to using glucose as the organic carbon source. This suggests that CSH is a better carbon source than glucose for heterotrophic Chlorella protothecoides.