Immobilized biomass of white rot fungus Trametes pubescens was explored for adsorption of azo dye Congo Red. The biomass immobilized by sodium alginate enhanced the sorption capacity for Congo Red approximately 4-fold of the free biomass, which competed well with other reported sorbents. Dye uptake was favored by acidic conditions at pH 2.0 with increasing initial dye concentration up to 100 mg/L using less biomass at room temperature and agitation speed. Adsorption of dyes onto the biomass was weakly dependent on ionic strength. Additionally, the adsorption process followed the pseudo-second-order kinetic and Freundlich isotherm models. During this process, the morphological changes on the biomass surface occurred; the amine functional groups present on the cell surface were mainly responsible for this process, and reduction in crystallinity of the biomass was observed, as confirmed by SEM, FT-IR, and X-ray diffraction, respectively. Desorption experiments were performed to regenerate the sorbent, making the process more economic and environmentally friendly. These demonstrated that the immobilized T. pubescens biomass is a promising sorbent for Congo Red removal.