The process of direct oxidation of penicillin G sulfoxide with penicillin fermentation broth was studied with peroxyacetic acid as oxidant, and a series of experiments were conducted to investigate the effects of different influencing factors on the conversion rate of penicillin G sulfoxide. The residual penicillin in the mycelium after oxidation was analyzed. The direct oxidation process of penicillin fermentation broth was established and optimized. The results showed that the stirring speed, reaction temperature, peroxyacetic acid feeding dosage and peroxyacetic acid concentration were the key factors affecting the conversion rate of penicillin G sulfoxide, other factors had little effect on the process. Peroxyacetic acid directly oxidized the penicillin fermentation broth and released penicillin remaining in the mycelium. So its conversion rate was higher than the conversion of penicillin G potassium salt. The optimum oxidation process conditions were reaction temperature of 5~10℃, stirring speed of 100 r/min, adding a high concentration of peroxyacetic acid in a molar ratio of 1.3 times of penicillin at 30 minutes, stirring reaction for 10 minutes after adding peroxyacetic acid. The conversion rate of penicillin G sulfoxide reached 98.6%, and the conversion rate increased by 1.2% than that of penicillin G potassium salt.