Due to the higher uncertainty of environmental risk for pollutants’ treatment by activated sludge, 10 mg/L influent 4‐chlorophenol (4‐CP) treated via a sequencing batch reactor (SBR) was used as acclimated SBR. Another SBR was used as control SBR without 4‐CP. The effects of hydraulic retention time (HRT) and proteins on sludge toxicity for 4‐CP treatment were analysed, and compared to the control SBR. Results showed that the sludge toxicity in acclimated SBR was significantly higher than that of the control SBR. Shortening HRT from 12 to 8 hours was beneficial to degrade 4‐CP and lower sludge toxicity. The identified highly expressed protein of ABC transporter co‐existed in control and acclimated SBRs, while other proteins of TonB‐dependent receptor, heat shock 70 kDa protein and superoxide dismutase in acclimated sludge were overexpressed relative to the control sludge, which played an important function in degrading 4‐CP, resisting 4‐CP toxicity and eliminating sludge toxicity.