We investigated optimal conditions for the hydrolysis of Laminaria japonica using a single enzyme such as Celluclast 1.5 L, Saczyme, and alginate lyase, for the production of reducing sugar. Redesigned experimental conditions including the optimal conditions determined for the single enzyme were proposed, and the hydrolysis of Laminaria japonica was also performed with a mixture of enzymes. The reducing sugar yield with the mixed enzymes was lower than that with Celluclast 1.5 L, which showed the highest efficiency among the enzymes used. Considering the reducing sugar yield and economics, it would seem that hydrolysis by mixed enzymes had no advantage. The coefficient of determination (R2) of Y1 (the yield of reducing sugar by Celluclast 1.5 L) was 0.89. The P value of Y1 was < 0.001, indicating statistical significance. By the response surface methodology (RSM), the optimum reaction conditions for hydrolysis of Laminaria japonica by Celluclast 1.5 L were determined to be enzyme of 8.0%, a reaction time of 26.4 h, a pH of 4.0, and a temperature of 42.6oC, resulting in the production of 117.7 mg/g-Laminaria japonica.