Abstract Olive tree pruning biomass is one of the main agricultural residues available in Mediterranean countries and is currently lacking commercial applications. To take advantage of its sugar content, a pretreatment is necessary to enhance enzyme accessibility of the cellulose fraction of the residue. This paper describes for the first time the use of organosolv pretreatment in this regard. The main process variables such as pretreatment temperature, residence time, and solvent composition (aqueous ethanol) are studied. Results show that organosolv pretreatment causes delignification and hydrolysis of hemicelluloses and improves the enzymatic digestibility of olive tree pruning biomass. A higher pretreatment severity and ethanol content of the solvent were found to increase delignification (up to 64% at 66% w/w aqueous ethanol, 210°C, 60 min). By contrast, xylan hydrolysis was promoted by a lower ethanol content (maximum 92%). The highest enzymatic hydrolysis yield (90% of the structural glucan present in the raw material) has been obtained after pretreatment with 43% w/w aqueous ethanol at 210°C for 15 min. Organosolv pretreatment was found to be the most effective pretreatment for enzymatic hydrolysis of olive tree pruning biomass.