This study investigated the optimal sequential hydrolysis conditions by comparing with reducing sugar yield ofsequential hydrolysis of Laminaria japonica processing residue. After acid-catalyzed hydrothermal hydrolysis, sequentialenzymatic hydrolysis was performed with single enzymes such as Celluclast® 1.5L, Saczyme, and Alginate Lyase, andtheir mixture. As a result, the yield of reducing sugar by sequential hydrolysis with the mixed enzymes was the highest,but there would be an economical problem with excessive enzyme loading. Therefore, considering the reducing sugaryield and economics, it is thought that hydrolysis by the mixed enzymes has no advantage, thus, using the Celluclast®1.5L in the sequential hydrolysis was practically more appropriate. The optimal sequential hydrolysis conditions ofLaminaria japonica processing residue were determined to be 8% v/w of enzyme injection, 42.6oC of reaction temperature,pH 4.1, and 26 hours of reaction time after acid-catalyzed hydrothermal hydrolysis (0.108 N-HCl, 144oC of reactiontemperature, and 22 minute of reaction time).