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Clostridium perfringens phospholipase C (plc), also called α-toxin, is encoded by the plc gene Clostridium perfringers. The production of α-toxin can lead to the occurrence of gas gangrene. Vaccination is considered as one of the best solutions against Clostridium infections. In this study, an anti-Cp α-toxin monoclonal antibody (mAbs) A10E5 was successfully prepared, which had better biological reactivity. Then, the phage random 12-peptide library was used to screen mAb A10E5 protein. After four rounds of screening, three peptides with high affinity to the anti-α-toxin mAbs were screened. Two 12-peptide peptide Q and peptide E with higher inhibition rate were obtained by indirect ELISA. Two polypeptides of 500 μg ? mL-1 synthesized in vitro were mixed with 30 μg ? mL-1 α-toxin at a concentration to treat Hela cells. Cell viability was determined by MTT assay. The results showed that both of the peptides significantly increased the survival rate of Hela cells compared with the α-toxin group, and the effect of peptide Q was more obvious. The chickens were immunized with phages expressing two different affinity polypeptides and then challenged. The results of chicken weight change, intestinal lesion score, bacterial count, and antibody titer in peripheral blood showed that the two phages expressing the polypeptides had a certain protective effect on the chickens compared with the PBS group, and peptide Q had better protection effect. In conclusion, the high affinity peptide with mAb A10E5 was screened in this study, and the protective effect of the plc polypeptide vaccine was verified by in vivo and in vitro experiments, which was of great significance for the comprehensive prevention and treatment of the disease.