Horseradish peroxidase (HRP) and glucose oxidase (GOx) were co-immobilized on polyurethane, and the resulting HRP/GOx/polyurethane biocatalyst was characterized using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDAX) mapping techniques. The prepared biocatalyst was used for removal of acid orange 7 as model azo dye. The required H2O2 for activation of HRP was in-situ produced using GOx to prevent deactivation of HRP in the presence of excess chemical H2O2. Central composite design (CCD) was applied for modeling and optimization of parameters affecting the activity of prepared biocatalyst. Under the optimum conditions, removal efficiency of the azo dye was predicted to be 87.47%, which was in good agreement with the experimental value (89.69%). In addition, the performance of the prepared biocatalyst for removal of two other dyes with different structure was investigated at the optimum conditions, and a removal efficiency of 91.56% and 95.25% was obtained for removal of methylene blue and malachite green, respectively. The results demonstrated that the resultant HRP/GOx/Polyurethane biocatalyst was able to decrease the chemical oxygen demand (COD) of a textile effluent from 740mg/L to 96mg/L, indicating that the prepared biocatalyst is an effective enzymatic system for treatment of real wastewater.