The fermentation residue in penicillin production was treated with Fenton reagent. The factors affecting penicillin removal were investigated and the reaction conditions were optimized by uniform design method. The results showed that the optimal conditions were as follows:pH=2,reaction temperature 69 ℃,reaction time 119 min and H_2O_2 dosage 0.6 mL/g. Under these conditions,the removal rate of penicillin in the residue reached over 99.77%,and most of nutrients in the residue were preserved,which provided t...