In this paper, the second generation pathway for bioethanol from hornbeam residues was investigated, in order to define the optimum operative conditions of steam explosion pretreatment and subsequently enzymatic hydrolysis maximizing the glucose amount for fermentation process. The whole experimental procedure was designed and analyzed by Response Surface Methodology, a statistic multivariate model that allows to investigate the effect of different parameters on a process and define the optimum values of these variables to optimize the response. The glucose yield from enzymatic hydrolysis was maximize as function of three variables: the severity factor of pretreatment (log R-0), the total solids (TS%) of the enzymatic hydrolysis and enzyme loading (EL %) in the enzymatic hydrolysis stage. The Design of Experiment (DoE), based on central composite model, was characterized by 17 tests, varying the variables at five levels, log R-0 (3.92, 4.08, 4.31, 4.54, 4.7), TS% (5, 7, 10, 13, 15), EL% (5, 7, 10, 13, 15). The optimization allows to define the best operative condition that is log R-0 = 3.97, TS% = 6, EL% = 15 which leads to an overall fermentable sugars yield of 67.8% respect to the initial sugars content in the raw material that corresponds to a theoretical amount of producible ethanol of 251 L/ton dry raw material. (C) 2017 Elsevier Ltd. All rights reserved.