Phanerochaete chrysosporium, a white rot basidiomycete, was immobilized over Luffa cylindrica sponge discs, treated with 0.1 N HCl and its potentiality for the removal of hexavalent chromium [Cr(VI)] from water was investigated in both batch and in up-flow fixed-bed bioreactor. The acid treatment of biomass increased the uptake capacity and percentage removal of Cr(VI) from 33.5 to 46.5 mg g−1 and 67 to 92 %, respectively. Maximum uptake of Cr(VI) was achieved at pH 2, temperature 40 °C after 100 min of contact time. The Cr(VI) sorption on the biomass was better explained by Langmuir isotherm. Thermodynamic studies indicated that the process was spontaneous and endothermic. Sorption kinetic study showed that pseudo-second-order model best correlates the Cr(VI) sorption on the biomass as compare to pseudo-first-order kinetic model. The performance of fixed-bed bioreactor was evaluated at different bed heights (5, 15 and 25 cm) and flow rates (1.66, 4.98 and 8.33 mL min−1) by using bed depth service time model. Response surface methodology statistical method was applied for optimizing the process parameters. FTIR analysis showed that amino groups were mainly involved in adsorption of Cr(VI).