The increasing demand for energy and the depletion of fossil fuel reserves have encouraged the development of renewable biofuels. Biobutanol is considered a promising alternative fuel because its physicochemical properties are more similar to gasoline than ethanol. In this study, biobutanol production from cornstarch was investigated through enzymatic hydrolysis followed by fermentation using Clostridium acetobutylicum. The hydrolysis performance was evaluated based on reducing sugar production, while the effects of enzyme concentration, surfactant addition, and hydrolysis time were examined. Tween-80 was used as a non-ionic surfactant to enhance the enzymatic hydrolysis process. The results showed that the addition of low concentrations of Tween-80 increased reducing sugar production, whereas excessive surfactant concentrations reduced hydrolysis efficiency. The optimum enzyme concentration for the liquefaction process was found to be 0.7% (v/v). Furthermore, increasing inoculum concentration initially enhanced biobutanol production; however, excessive inoculum concentrations led to decreased butanol production due to nutrient limitation and fermentation inhibition. These findings demonstrate the importance of optimizing hydrolysis and fermentation conditions to improve biobutanol production from cornstarch.