© 2021 Hydrogen Energy Publications LLCThis study investigated the best pretreatment conditions for olive tree biomass (OTB) to produce bio-hydrogen. The pretreatment conditions were optimized with response surface methodology for the dark fermentative hydrogen production to achieve maximum reducing sugar recovery and the independent variables were residence time (30–90min), temperature (100–140 °C), and acid concentration (0.5–3 %w/w for sulfuric and 5–10 %w/w for oxalic acid). Maximum reducing sugar obtained after sulfuric acid pretreatment and oxalic acid pretreatment at optimal pretreatment conditions were 37 g/L and 28 g/L, respectively. An approximately 20-fold increase in sugar concentration was achieved in pretreatment with sulfuric acid compared to the untreated control. The highest hydrogen yield was 0.83 and 0.91 mol H2/mol reducing sugar for oxalic acid pretreatment and sulfuric acid pretreatment, respectively. The results show that OTB has the potential to be used in bioenergy production.