Microalgae are considered promising feedstocks for biofuel and bio-product generation. The algal carbohydrates can be hydrolyzed into sugars before their fermentation into ethanol. In this study, nutrient limitation strategy was employed to evaluate the biochemical composition of Chlorella sorokiniana. Limiting nitrate (1.0 g/L KNO3) in the culture medium increased the total carbohydrate and starch content of microalga by 50.28 and 34.06%, respectively. However, this significantly lowered their yield due to low microalgal biomass production. Cultivation of C. sorokiniana cells with 4.0 g/L KNO3 as nitrogen source for 8 days was optimum for bioethanol production as the highest total carbohydrate yield of 422.44 mg/L was obtained under these conditions. Nitrate limitation (1.0 g/L KNO3) favored the increased production of high-value carotenoids in C. sorokiniana that could further contribute to improving the economics of the bioethanol production process. Feasibility studies for ethanol production from C. sorokiniana revealed that a maximum of 13.86 mg/mL of reducing sugars was extracted in the hydrolysate by treating the microalgal biomass with 2.8% sulfuric acid at 121 °C for 30 min. Fermentation of acid hydrolysate produced ethanol at a concentration of 2.91 mg/mL in 96 h with 41.16% of theoretical yield.