Abstract This study describes the characterization of the recombinant GH3 aryl-β-glucosidase “GluLm” from Limosilactobacillus mucosae INIA P508, followed by its immobilization on an agarose support with the aim of developing an efficient application to increase the availability and concentration of flavonoid and lignan aglycones in a vegetal beverage. In previous studies, heterologous GluLm-producing strains demonstrated a great capacity to deglycosylate flavonoids. Nevertheless, the physicochemical properties and substrate spectrum of the enzyme remained unknown up to now. A high production of purified GluLm was achieved (14 mg L⁻¹). GluLm exhibited optimal activity at broad ranges of pH (5.0–8.0) and temperature (25–60°C), as well as high affinity (K_m of 0.10 mmol L⁻¹) and specific constant (86554.0 mmol L⁻¹ s⁻¹) against p-nitrophenyl-β-D-glucopyranoside. Similar to other GH3 β-glucosidases described in lactic acid bacteria, GluLm exhibited β-xylosidase, β-galactosidase, and β-fucosidase activities. However, this study has revealed for the first time that a GH3 β-glucosidase is capable to hydrolyze different families of glycosylated phenolics such as flavonoids and secoiridoids. Although it exhibited low thermal stability, immobilization of GluLm improved its thermostability and allowed the development of a beverage based on soybeans and flaxseed extract with high concentration of bioactive isoflavone (daidzein, genistein), lignan (secoisolariciresinol, pinoresinol, and matairesinol), and other flavonoid aglycones. Key points • Limosilactobacillus mucosae INIA P508 GluLm was purified and biochemically characterized • Immobilized GluLm efficiently deglycosylated flavonoids and lignans from a vegetal beverage • A viable application to produce vegetal beverages with a high content of aglycones is described