This study investigated the effect of alkaline treatment and various acid hydrolysis conditions on the extraction of cellulose nanofibers from sugarcane bagasse. Alkaline treatments were conducted using sodium hydroxide and sodium hypochlorite solutions. After the alkaline treatment, acid hydrolysis was performed in solutions containing 60, 64, and 68% wt. of sulfuric acid at temperatures of 30 ± 1 °C and 45 ± 1 °C. The effectiveness of the alkaline treatment in removing non-cellulosic components was assessed by infrared spectrometry and scanning electron microscopy, which demonstrated satisfactory results in eliminating non-cellulosic components. Transmission electron microscopy revealed the impact of temperature on the dimensions of the extracted cellulose nanofibers, with acid hydrolysis at 45 °C yielding smaller nanofibers compared to hydrolysis at 30 °C. X-ray diffraction analysis showed a higher percentage of crystallinity for hydrolysis at 45 °C, which can be attributed to the removal of a substantial amount of amorphous cellulose content.