In the present study, a novel laccase from ascomycete Gliomastix murorum was produced in agro-industrial wastes and entrapped in galactomannan beads for Reactive Blue 2 (Rb-2) decolorization. The maximum laccase production in agave bagasse-based medium occurred at 72 h (1798.6 UL⁻¹). Entrapped laccase decolorized ˃80% of 0.5 mM Rb-2 in 2 h without the addition of redox mediator. K_m for Rb-2 substrate was 1.42 mM, with a V_max of 1.19 µmol min⁻¹ for entrapped laccase. Galactomannan matrices produce stability to acid pH (2–5) and temperatures from 20–70 °C. Reusability assays showed that entrapped laccase could retain efficient Rb-2 decolorization of ˃80% six times. In general, galactomannan used for entrapment of laccase provides economic advantages in large-scale wastewater treatment due to its natural origin and efficient results.