This study investigated the effect of sequential oxidative (ozonolysis) pretreatment and enzymatic hydrolysis of hazelnut shells on production of fermentable sugars. Two factors (ozone concentration and pretreatment time), which were determined to be significant by the Box-Behnken response surface methodology, were further maximized for total lignin removal, solid recovery, and total reducing sugar yield via enzymatic hydrolysis of the pretreated biomass using full-factorial design. The ozonolysis was performed by passing 30, 40, and 50 mg/L of ozone gas through a packed bed of ground hazelnut shells for up to 120 min at a flow rate of 0.25 L/min. The highest lignin reduction (20.5%) was obtained in the shells with 30% moisture content subjected to ozone concentration of 50 mg/L for 120 min, which corresponded to a high biomass recovery (94%). The reducing sugar yield also increased from 119.9 mg/g dry untreated biomass to 284.6 mg/g dry untreated biomass for hazelnut shells ozonated at 30 mg/L for 60 min. Thus, these results indicate remarkable benefits of ozonolysis during pretreatment of hazelnut shells.