The pretreatment of Helianthus tuberosus residue was studied for fermentable sugar production. The pretreatment was performed by varying the temperature, type of chemical solution, and concentration. Two different catalytic pretreatments using sulfuric acid and aqueous ammonia were operated and compared in a flow-through column reactor system. The flow-through process was required to increase the sugar production yield of biomass. To selectively remove the lignin of biomass and achieve fractionation of hemicellulose in the liquid phase to produce pentose, the flow-through process could be controlled by the pretreatment conditions. Furthermore, the remaining solid underwent enzymatic hydrolysis for hexose production. The mass balances of biomass pretreated with aqueous ammonia and sulfuric acid solution were compared in terms of production of fermentable sugars. The glucose recovery compared to the initial biomass was 71.2% in the pretreatment using aqueous ammonia at 170 °C, and pretreatment using sulfuric acid solution at 150 °C was 52.3%.