Human papillomavirus E7 gene is the key to cancer, which expressed in most HPV-contains cervical cancers. E7 plays an important role in the induction and maintenance of cellular transformation, and it was regarded as the ideal target in cervical cancer treatment. An important development direction of cervical cancer therapy is based on HPV16 E7 antigen CTL epitope peptide vaccine design, but Short half-life in vivo and poor stimulation effect are the major therapeutic obstacles for native CTL epitope peptide. In this study, three epitope peptides were screened by molecular dynamics simulations with MM-PBSA method, associated with previous research results and polypeptide enzymolysis experiment. These predicted peptides were synthesized, examining the affinity between HLA-A2 molecule and each peptide by T2 cell line. The result showed synthesized peptides had a great improvement on enzyme-resistant ability comparing with natural HPV16 E7 CTL epitope antigen peptide, and (d)RAHYNIVTF was the most obvious epitope peptide. Meanwhile, the affinity was improved between (d)RAHYNIVTF and HLA-A2 (fluorescence indexes was 2.06). The method of structural modification in this study is expected to lay the foundation for cervical cancer therapeutic vaccine design.