Background: β-Glucosidase is known as an effective catalyst for the hydrolysis of various glycosides and immobilization is one of the most efficient strategies to improve its activity recovery and properties. Results: Crosslinking-adsorption-crosslinking method was employed to immobilize β-glucosidase into chitosan beads and response surface methodology (RSM) was used to optimize the immobilized conditions of the maximum activity recovery. Enzyme concentration and adsorption time were found to be significant influence factors, and the maximum activity recovery (50.75%) obtained from response surface methodology was in excellent agreement with experimental value (50.81%). Furthermore, various characteristics of immobilized β-glucosidase were evaluated. Compared to the free β-glucosidase, the immobilized enzyme exhibited broader pH and temperature ranges, enhanced thermal stability, better storage stability and reusability and higher accessibility of the substrate to the immobilized β-glucosidase. Conclusion: Response surface methodology (RSM) was proved to be much economical for optimum immobilization of β-glucosidase into chitosan beads.