© 2021 Elsevier LtdCellulase activity is a critical parameter for generating alternative renewable fuels. For observing this activity, Avicel (microcrystalline cellulose) is a widely used substrate. As lignocellulosic biomass in nature, peanut hull (35% cellulose) is a suitable material to convert the chemical feedstocks. Alkaline pretreatment (with NaOH solution at 121 °C for 1 h) is a necessary process to increase the yield of the hydrolysis due to the high lignin content of the biomass. The objective of this study is to determine the effects of high hydrostatic pressure (HHP) on cellulase-hemicellulase (Celluclast) activity and the subsequent hydrolysis. Enzyme mixtures were subjected to 100 and 500 MPa for 5–15 min, at 30 °C both with and without substrate. HHP-treated enzyme and enzyme-substrate solutions had significantly (p < 0.05) higher reducing sugar content (up to 2.06 and 2.83 g/L increases for Avicel, 0.85 and 0.96 g/L for peanut hulls; respectively) than non-HHP-treated solutions. Nevertheless, no significant difference was observed between 100 and 500 MPa (p > 0.05). NMR T2 relaxation times of the treated and hydrolyzed samples were affected by HHP. Additionally, enzymatic hydrolysis significantly increased the T2 (p < 0.05). The changes on peanut hulls after HHP, alkaline pretreatment, and enzymatic hydrolysis were observed by using FTIR spectrometry.