A response surface methodology (RSM) tool with the Box-Behnken design was used to determine the optimum pretreatment conditions of Helianthus tuberosus residue for the enzymatic production of fermentable sugar with aqueous ammonia and sulfuric acid solutions, for various parameters such as pretreatment solution concentration, temperature, and reaction time. The pretreatment of biomass was performed using these optimized parameters in aqueous ammonia and sulfuric acid solution, followed by hot water, under the same conditions. The process was then performed by changing the sequence.