The lignocellulosic biomass, such as provided by the sugarcane, is an abundant source of raw materials for energy production. Milling and pretreatments can be employed to alter the structure of the materials, remove lignin, and hemicellulose. This pretreatment effect exposes the cellulose and raises its accessibility, which is one of the most important properties to ensure enzymatic digestibility. However, the biomass generated from the sugarcane has different physicochemical characteristics, giving different responses to the pretreatments. In this context, this study aimed to verify the effects of lignin and hemicellulose removal from the sugarcane biomass (external fraction, node, internode, and leaf) on cellulose accessibility. Each fraction was pretreated with acid (5, 10, and 20\% w/w, at 121 degrees C/30 min), alkali (5, 10, 20, and 30\% NaOH w/w) and oxidative (0.5, 1, 2, and 3 h charged with 30\% sodium chlorite). Accessibility was determined by dye adsorption of Direct Orange (external specific surface) and Direct Blue (internal specific surface). Enzymatic hydrolysis was used to verify the effects of pretreatments and cellulose accessibility on the glucose yield. Delignification by sodium chlorite (oxidative) resulted in lignin removal, with almost complete removal from leaf samples. Accessibility determined indicated that pretreatments that are more aggressive improved cellulose accessibility. The less recalcitrant fraction, the internode, showed 1333.3 mg/g of Direct Orange adsorbed and 746.3 mg/g of Direct Blue. Glucose yield during enzymatic hydrolysis improved with higher cellulose accessibility. Lignin and xylan removal (down to 10\% and 1\%, respectively) resulted in higher glucose yield, with delignified internode samples showing almost complete cellulose conversion. Hemicellulose and lignin removal by the pretreatments directly influenced cellulose accessibility, resulting in better enzymatic hydrolysis across all fractions. This study successfully showed that lignin and hemicellulose removal of 15\% and 10\%, respectively, resulting in at least 60\% of glucose yield, reaching desired accessibility levels based on dye adsorption of 2079.6 mg/g.