In this study, three indigenous microalgae strains (Scenedesmus subspicatus GY-16, Chlorella vulgaris FSP-E, and Anistrodesmus gracilis GY-09) were evaluated for their performance on producing carbohydrates, which serve as feedstock for bioH2 production. The results show that C. vulgaris FSP-E displayed the highest growth rate (825.6 mg/L/d) and carbohydrate productivity (365.8 mg/L/d). Different sodium acetate concentrations were supplemented into the growth medium to investigate the effect of organic carbon source on biomass and carbohydrate productivity. The results show that using 2000 mg/l sodium acetate to grow C. vulgaris FSP-E resulted in the highest biomass and carbohydrate productivity of 1022.3 mg/l/d and 498.5 mg/l/d, respectively. Meanwhile, to assess the practical applicability of the proposed microalgae-based carbohydrates production system, the photobioreactor was further operated on semi-batch mode for a prolonged incubation time under the optimal conditions. The results show that the biomass and carbohydrate productivity of C. vulgaris FSP-E was stably maintained at 1173.6 and 371.4 mg/L/d, respectively. Due to the high carbohydrate content, C. vulgaris FSP-E is considered an excellent feedstock for biohydrogen fermentation. Using the acidic hydrolysate of C. vulgaris FSP-E as feedstock, the separate hydrolysis and fermentation (SHF) process could achieve high-yield hydrogen production with a maximum hydrogen yield of 2.67 g hydrogen/g microalgae biomass, which is higher than most reported values. These results indicate that using carbohydrate-rich microalgae as feedstock to produce biohydrogen is indeed feasible for practical applications.