Abstract Peanut shells (i.e., an abundant industrial by-product) were subjected to an innovative hydrothermal pretreatment approach using high-pressure CO2 to enhance their enzymatic hydrolysis conversion into glucose. This pretreatment led to a reduction in hemicellulose content in the pretreated peanut shells from 12.4% to as low as 1.8%, which facilitated subsequent conversion into glucose by enzymatic hydrolysis. This pretreatment approach was assessed within a 170–200 °C temperature range and a 20–60 bar CO2 pressure range, after which the results of these conditions were compared to those of conventional hot water pretreatment. Treatment at 190 °C and a 60-bar CO2 pressure was determined to be optimal, resulting in the highest glucose yield (80.7%) from subsequent enzymatic hydrolysis. Acidic conditions resulting from CO2-derived carbonic acid significantly reduced the hemicellulose content of the peanut shells and weakened the interaction between cellulose, hemicellulose, and lignin, improving enzyme accessibility to the cellulose. Furthermore, high-pressure CO2 increased the pore size and porosity of the resulting pretreated peanut shells, improving their enzyme adsorption capacities, as confirmed by cellulase adsorption and mercury intrusion porosimetry tests. The dual effect from high-pressure CO2 led to significant hemicellulose reduction and improved adsorption of enzymes on the cellulose, which in turn increased glucose yield from the subsequent enzymatic hydrolysis of pretreated peanut shells. Alcoholic fermentation of the hydrolyzed glucose resulted in a 12.4% increase in bio-ethanol production compared to a glucose control, thus highlighting the potential of pre-treated peanut shells as a glucose precursor used in biofuel industry.