Biomass pretreatment for the production of second-generation (2G) ethanol and biochemical products is a challenging process. The present study investigated the synergistic efficiency of purified carboxymethyl cellulase (CMCase), β-glucosidase, and xylanase from Aspergillus fumigatus JCM 10253 in the hydrolysis of alkaline-pretreated sugarcane bagasse (SCB). The saccharification of pretreated SCB was optimised using a combination of CMCase and β-glucosidase (C + β; 1:1) and addition of xylanase (C + β + xyl; 1:1:1). Independent and dependent variables influencing enzymatic hydrolysis were investigated using response surface methodology (RSM). Hydrolysis using purified CMCase and β-glucosidase achieved yields of 18.72mg/mL glucose and 6.98mg/mL xylose. Incorporation of xylanase in saccharification increased the titres of glucose (22.83mg/mL) and xylose (9.54mg/mL). Furthermore, characterisation of SCB biomass by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy respectively confirmed efficient structural disintegration and revealed the degree of crystallinity and spectral characteristics. Therefore, depolymerisation of lignin to produce high-value chemicals is essential for sustainable and competitive biorefinery development.