Microalgal carbohydrates can serve as feedstock for bioethanol production. Environmental and/or nutritional factors influence the carbohydrate content of microalgae and optimization of culture condition has a key role in the economic production of bioethanol. This research has evaluated the optimization of the carbohydrate content of a marine microalga isolate from the Persian Gulf. The Plackett-Burman design was used to screen the most effective parameters responsible for carbohydrate production. Then, the levels of two effective factors, total phosphate (KH2PO4þK2HPO4) and initial pH, were optimized by two types of Response Surface Method designs (Central Composite and historical data designs). Maximum carbohydrate production was obtained at a low concentration of phosphate (0.031 g/L) and initial pH of 5.95. Under optimal condition, the concentration of carbohydrate was increased by 1.9 folds, from 0.12 g/L (0.2 g/g of dry biomass) to 0.230 g/L (0.365 g/g of dry biomass). The carbohydrate content of microalga hydrolysate was also evaluated by the HPLC method, and the results showed that glucose made about 3.19% (w/w) of total carbohydrates. This study represents the isolation and determination of a promising native and non-invasive microalga for bioethanol production, and subsequently enhancement of its carbohydrate content for the same goal.