Enzymes can be used in various biotechnological applications due to the easy and cheap production. Since xylanase enzymes are preferred in various industries, researchon this enzyme is extensively being carried out. In this study, the β-xylanase gene was cloned from Thermotoga naphthophila, a thermophilic organism. The expression vector pET21a(+) was expressed in Escherichia coli BL21 (DE3). As a result of the studies, the pH, temperature and IPTG concentration of the enzyme were optimized to obtain highest expression. Dinitrosalicylic acid (DNS) was used to determine sugar content of the enzyme. The molecular mass of the purified β-xylanase enzyme was determined using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular mass of the enzyme was calculated to be 38 kDa. Enzymatic hydrolysis of hazelnut shell, rhododendron branch and rhododendron leaves was performed. Released reducing sugar contents from the enzymatic hydrolysis were calculated as 0.8461 mg mL-1, 0.6976 mg mL-1 and 0.3605 mg mL-1 for hazelnut shell, rhododendron branch, and rhododendron leaf respectively. In conclusion, β-xylanase enzyme can be an effective source for enzymatic hydrolysis to produce fermentable sugars from such biomasses.