Microalgae can grow heterotrophically with glucose as a carbon source to accumulate high amounts of lipids, which are one of the most promising feedstock for biodiesel production. However, glucose is one of the main contributors in the high cost of microalgae culture. This study was to evaluate the feasibility of using potato starch hydrolysate (PSH) instead of glucose as a carbon source for microalgal lipid production. When PSH was used as a carbon source for the heterotrophic microalga Chlorella protothecoides, the highest biomass and total lipid yields were 20.23 and 10.43 g/L, respectively, representing 1.3- and 1.4-fold higher than those obtained when glucose is used as a carbon source. A noticeable increase in the number of oil droplets was observed in the PSH-feeding algal cells. In addition, the calorific value was significantly increased to 29.08 kJ/g compared to the calorific value of 22.65 kJ/g from the cells grown in glucose medium. These findings confirmed that the lipid content greatly improved in PSH-feeding cells. Moreover, PSH-feeding cells produced significantly elevated levels of 17:0, 17:2(Delta 9,12), 19:1(Delta 9), and 19:2(Delta 9,12) fatty acids (over 84%), which are feasible ingredients for biodiesel production. These results demonstrated that green alga fatty acids from heterotrophic C. protothecoides obtained by PSH feeding possess properties that are beneficial for biodiesel production.