Bioethanol is produced from lignocellulosic materials, such as agricultural waste, forest residues, etc. In this study, cotton gin waste was utilized to produce fermentable sugars through an effective pretreatment method. Autoclaving, steam explosion, and liquid hot water pretreatments were compared for their effectiveness in disruption of the lignocellulose matrix. Autoclaving was proved to be an effective pretreatment method in terms of maximum reducing sugar production. The Box-Behnken design (BBD) was employed in the optimization of autoclaving pretreatment. Slurry density (X-1, g/mL), temperature (X-2, degrees C), and time (X-3, min) were chosen as the independent variables and the total reducing sugar concentration (Y, mg/L) as the dependent variable (response). The total reducing sugar production from cotton gin waste was found to be 511.48 mg/L experimentally, at an optimum slurry density of 0.50 g/mL, a temperature of 138 degrees C, and a time of 11 min.