A β-glucosidase gene from Paenibacillus barengoltzii CAU904 was cloned and heterologously expressed in E. coli. The recombinant enzyme was purified and biochemically characterized, and its crystal structure was further resolved. The enzyme had the highest activity at 50℃ and pH 7.5. It exhibited broad substrate specificity on various substrates, including laminarin, barley β-glucan, lichenan, et al. (containing β-1,2,β-1,3,β-1,4 and β-1,6 linkages). Crystal structure analysis revealed that the enzyme exhibited typical glycoside hydrolyase family 3 enzymes, having multiple domain structures. The catalytic packet of the enzyme was composed by a (α/β)₅ sandwich domain and a (β/α)₈ folding barrel domain linked by a loop region. The side chain aromatic amino acids Trp748 and Trp749 were functioned as -1 and +1 binding site, respectively, while Trp131 was functioned as +2 site. These aromatic amino acids formed a narrow pocked and a hydrophobic environment, which could diminish its transglycosylation activity.