In this study, the pretreatment of Helianthus tuberosus residue had been performed. The two-stage pretreatment on flow-through process were applied in the interests of increase of sugar production yield on enzymatic saccharification. The delignification by aqueous ammonia and the fractionation of hemicellulose by sulfuric acid solution as pretreatment solution were confirmed for effects of enzymatic saccharification. Two-stage pretreatment process was performed using aqueous ammonia and sulfuric acid. The first step was performed with aqueous ammonia for 40 min at 163.2 oC and the second step was performed with sulfuric acid solution for 20 min at 169.7 oC. And then, the first step was performed with sulfuric acid solution and the second step was pretreated with aqueous ammonia. At this time, the glucose production was 30.7 g and the glucose yield was 72.4% in the first step process with aqueous ammonia. And, the glucose production was 20.9 g and the glucose yield was 49.3% in the first step process with sulfuric acid solution.