The paper aims to study the interactive mechanism of alisol B 23-acetate (23B), alisol A 24-acetate(24A) and content of 23B∶24A=1∶1 mixture with p53DNA which is tumor-suppressor gene, and explore the molecular mechanism of the antitumor effects of alisol acetates. The interaction of 23B, 24A and 24A-23B mixture with p53DNA was investigated by Ultraviolet and Visible Spectroscopy (UV-Vis), Fluorescence spectroscopy and Molecular simulation. UV-Vis showed that p53DNA was partially inserted by alisol mononers and its mixture, in which the decrease degree between p53DNA’s UV-Vis absorbance was 24A∶23B (1∶1)>23B>24A. Fluorescence spectroscopy demonstrated that the mode of interaction of p53DNA and alisol mononers and its mixture was inserted, in which the bonding strength was 24A∶23B (1∶1)>23B>24A. Molecular simulation illustrated that the sequence of binding of alisol mononers and its mixture with p53DNA was 24A∶23B (1∶1)>23B>24A, in which 23B formed a hydrogen bond with Adenine nucleotides (DA4) of p53DNA while 24A-23B mixture formed four hydrogen bonds with the DA4 and Thymine Nucleotides (DT19). The sequence of binding of alisol mononers and its mixture with p53DNA was 24A∶23B(1∶1)>23B>24A, in which the mode of interaction between them and p53DNA were all partial insertion, indicating that 24A and 23B had synergistic effects on anticancer target p53DNA. In addition, the parental rings C14- of alisol acetates and their steric hindrance and the oxygen in phosphoric acid of DA4 in the f chain in p53DNA were the binding site of the interaction between alisol acetates and p53DNA, which was the active center of the antitumor effects of alisol acetates. The key to the synergy of alisol acetates is that the the hydroxyl group on the side chainn C19- of 24A and the nitrogen and oxygen on adenine of DA4 in the f chain in p53DNA and the oxygen atom in thymine of DT19 in the e chain in p53DNA.