Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.