This study investigated the production of fermentable sugars from carbohydrate-rich macroalgae Saccharina japonica using sequential hydrolysis (hydrothermal acid pretreatment and enzymatic hydrolysis) to determine the maximum reducing sugar yield (RSy). The sequential hydrolysis was predicted by three independent variables (temperature, time, and HCl concentration) using response surface methodology (RSM). Enzymatic hydrolysis (8.17% v/wbiomass Celluclast® 1.5 L, 26.4 h, 42.6 °C) was performed after hydrothermal acid pretreatment under predicted conditions (143.6 °C, 22 min, and 0.108 N HCl concentration). Using this experimental procedure, the yields of hydrothermal acid pretreatment, enzymatic hydrolysis, and sequential hydrolysis were 115.6 ± 0.4 mg/g, 117.7 ± 0.3 mg/g, and 183.5 ± 0.6 mg/g, respectively. Our results suggested that sequential hydrolysis of hydrothermal acid pretreatment and enzymatic hydrolysis was more efficient than their single treatment.