Olive tree biomass (OTB) represents an interesting feedstock for bioethanol production. In this study, olive tree pruning was water extracted and pretreated by dilute sulfuric acid to achieve high sugar recoveries from cellulosic and hemicellulosic fractions. Temperature (160 to 200 °C), acid concentration (0 to 8 g acid/100 g extracted raw material), and solids loading (15% to 35% w/v) were selected as operation variables and modified according to a Box-Behnken experimental design. The optimal conditions for the acid pretreatment were 160 °C, 4.9 g sulfuric acid/100 g biomass, and 35% solids loading (w/v), according to multiple criteria that considered the maximization of both the hemicellulosic sugars concentration in prehydrolysate and the overall sugar yield. These optimized conditions yielded a sugar concentration of 79.8 g/L, corresponding to an overall yield of 39.8 g total sugars/100 g extracted OTB. The fermentability of hemicellulosic sugars prehydrolysates from the acid pretreatment was evaluated by Escherichia coli after a detoxification stage by overliming. The prehydrolysates with lower concentrations of toxic compounds were fermented and achieved ethanol yields higher than 80% of the theoretical ethanol yield.