2,4-Dichlorophenol (2,4-DCP) acclimated activated sludge culture was immobilized in polyurethane foam and supplemented to a packed upflow column bioreactor (PUCB) to treat 2,4-DCP containing wastewater. The impact of sludge age and 2,4-DCP concentration on biodegradation performance were studied. Results show that sludge age affected biodegradation in the PUCB. The residence time was adjusted by feeding with synthetic wastewater without DCP. The optimum phenol concentration was determined in 5-, 10-, and 15-day-old sludge. According to the results obtained, the removal of 99% phenol and 95% COD were determined in 10-day-old sludge and 500 mg/L 2,4-DCP concentration. Packing medium was examined both visually and by SEM, and a significant amount of organisms was observed accumulating on the surface, which indicated that diffusion limitations were controlling biofilm thickness.