Glucose tolerant β-glucosidases have attracted considerable attention due to their important roles in various biotechnological processes. Penicillium sp. NRC24, a new fungal isolate was used to produce soy glycosides-hydrolyzing β-glucosidase. The ability of β-glucosidase to convert soy glucosides into aglycone forms, as well as its activity on p-NPG, was investigated. Penicillium sp. NRC24 β-glucosidase was partially purified with 8.04-fold and final recovery of 45.7%. This enzyme exhibited a glucose-tolerant activity of 60.4% while the partially purified enzyme showed 85.6% activity at 0.5 M of glucose. Moreover, the produced β-glucosidase could tolerate ethanol up to 60%. β-glucosidase retained all its initial activity at 37 °C in presence of 15-60 % ethanol at pH 5.0. The immobilization and characterization of the produced β-glucosidase are described. The efficacy of β-glucosidase covalently immobilized on alginate/polysorbate complex was 70.1%. The impact of pH, and thermal stability, on the free and immobilized β-glucosidase activities and hydrolysis of soybean extract were evaluated. The optimum pH remained at 5.0 after immobilization, while the optimum temperature leveled up to 50 °C in comparison to the free enzyme, which remained at 37 °C. This is the first report to our knowledge on the use of Penicillium sp. NRC24 for the production, characterization, and immobilization of β-glucosidase, which can hydrolyze genistein and daidzein into their aglycone forms.