Oil palm empty fruit bunches (EFB) and mesocarp fibers (MF) are residual materials from the palm oil industry, distinguished by their notably high levels of cellulose. These lignocellulosic residues serve as valuable raw materials due to their substantial cellulose proportion, making them suitable for various bioconversion processes. In this study, ozonolysis pretreatment was employed to concentrate the cellulose present in EFB and MF, while simultaneously removing lignin content. Subsequently, the cellulose was hydrolyzed enzymatically with cellulase to produce reducing sugars. The contents of cellulose, hemicellulose, and lignin in both raw materials and samples were analyzed through the α-cellulose, γ-cellulose, and Kappa methods, while reducing sugars were quantified using the DNS (3,5-dinitrosalicylic acid) assay. The objective was to evaluate the relative effectiveness of ozonolysis as a pretreatment and to examine how enzyme concentration and reaction duration influence substrate conversion and sugar production from EFB and MF. This analysis provides valuable insight into optimizing the enzymatic hydrolysis process for these biomass materials. Results revealed a progressive increase in cellulose composition for both substrates, with MF showing a more pronounced response than EFB. The reducing sugar content for EFB and MF samples with varying enzyme concentrations increased continuously, reaching an optimum at 42 h. The maximum reducing sugar concentration for EFB was 0.1976 mg/mL, obtained with 15% (w/w) cellulase enzyme (Novozyme), whereas for MF, the highest reducing sugar concentration was 5.23 mg/mL with 10% (w/w) cellulase enzyme treatment.