The study on “Purification and characterization of endoglucanase from Bacillus subtilis S2O” was carried out to establish a purification procedure for endoglucanase and determine its characters. Broth incubated with Bacillus subtilis S2O at 38oC in an anaerobic condition for 5 days was collected and then centrifuged to remove bacterial cells and substrate. Endoglucanase was precipitaed by ammonium sulfate and purified by ion exchange chromatoghaphy on Uno Sphere Q gel. The fraction of 70% and 80% saturation of ammonium sulfate exhibited the highest specific activity, 105 and 119 (U/mg), respectively. These fractions were further purified in next step. The F1 fraction, eluted by 0.12M NaCl on Uno Sphere Q gel, had the highest specific activity of 720 U/mg and 0.24 mg protein in total. The yield and purity level were 24.3% and 23, respectively. The purified endoglucanase can be two isozymes with molecular weight of 79.6 kDa and 74 kDa. The purified enzyme had optimum temperature at 60oC and optimal pH 8. Endoglucanase activity was activated by K+ ion up to 148% when compare to the control treatment (100%) and was inhibited by Mn2+, Fe3+ and Cu2+. Endoglucanase exhibited highest activity against CMC. However, it also had ability to hydrolyze crystalline forms of cellulose such as avicel and filter paper but it was too low.