The function and application of β-glucosidase attract attention nowadays. β-glucosidase was confirmed of transforming ginsenoside Rb to rare ginsenoside, but the interaction mechanism remains not clear. In this work, β-glucosidase from GH1 family of Paenibacillus was selected, and its gene sequence was synthesized by codon. Then, recombinant plasmid was transferred into BL21 (DE3) and expressed. The UV-visible spectrum showed that ginsenoside Rb decreased the polarity of the corresponding structure of hydrophobic aromatic amino acids (Trp) in β-glucosidase and increased new π-π transition. The fluorescence quenching spectrum showed that ginsenoside Rb inhibited intrinsic fluorescence, formed static quenching, reduced the surface hydrophobicity of β-glucosidase, and K was 8.37 × 10 L/M (298K). Circular dichroism (CD) showed that secondary structure of β-glucosidase was changed by the binding action. Localized surface plasmon resonance (LSPR) showed that β-glucosidase and Rb had strong binding power which KD value was 5.24 × 10 (±2.35 × 10) M. Molecular docking simulation evaluated the binding site, hydrophobic force, hydrogen bond, and key amino acids of β-glucosidase with ginsenoside Rb in the process. Thus, this work could provide basic mechanisms of the binding and interaction between β-glucosidase and ginsenoside Rb.