The microalgae have cellulose as a main structural component of their cell wall and the lignin content in microalgae is much lower than other lignocellulosic biomass. Therefore, fermentable sugar production from microalgae (Tetraselmis KCTC 12236BP) can be carried out under pretreatment without high temperature and high pressure. It was investigated that the effect of hot-water pretreatment using sulfuric acid for lipid extracted algae which is expected to be a next generation biomass. The effects of three major variables including extraction temperature, acid concentration and time on the enzymatic hydrolysis were investigated. Among the tested variables, temperature and acid concentration showed significant effects and optimum pretreatment conditions for the economic operation criteria were obtained as follows: reaction temperature of 120 ℃, sulfuric acid concentration of 2 mol and pretreatment time of 40 min. Under the optimum conditions of acidic hot water pretreatment, experimentally obtained hydrolysis yield were 95.9% which showed about 2.1 fold higher compared with enzymatic hydrolysis process. Therefore, acid pretreatment under mild condition was proven to be an effective method for fermentable sugar production from lipid extracted microalgae.