(1,3)(1,4)-beta-D-glucan has been determined to have various beneficial effects due to its unique structure. beta-glucooligosaccharides (beta-GOS), which are hydrolysates of barley (1,3)(1,4)-beta-D-glucan, provide a useful pre biotic material for selective growth of probiotic bacteria. In this study, recombinant beta-1,3-1,4-glucanase (Bg1314) from Bacillus sp. SJ-10 (KCCM 90078) was immobilized on porous silica using glutaraldehyde as a crosslinking reagent to achieve efficient production of beta-GOS. We investigated the effects of factors such as the amounts of enzyme and glutaraldehyde, reaction temperature, and pH on catalytic activity. Enzyme activity decreased sharply at high concentrations of glutaraldehyde, likely due to the reaction of glutaraldehyde with lysine residues at the catalytic site of Bg1314, because lysine-substituted Bg1314 retained its activity under the same conditions. Immobilized Bg1314 protein (ImBg1314) was stable over a wide range of pH and could be stored long term at 4 degrees C. The optimal conditions of ImBg1314 were similar to those of Bg1314. However, the optimal temperature of ImBg1314 differed from that of Bg1314. The products were beta-GOS composed of 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose. After ImBg1314 was reused for 10 cycles, it retained 42% of its initial catalytic activity. This study showed that the Imbg1314 applied economical production of beta-GOS.