p53 is a pro-apoptotic tumor suppressor. As a transcription factor, p53 directly regulates a variety of cellular processes, including apoptosis, cell cycle, cell senescence, metabolism, and antioxidation. Azurin is a copper-containing redox protein secreted by Pseudomonas aeruginosa. It consists of 128 amino acids. Azurin has attracted much attention because it preferentially enters a variety of cancer cells and produces cytotoxicity. p28 is a segment of azurin, corresponding to residues 50 to 77. So far, several different mechanisms have been proposed to explain the anticancer activity of azurin and p28, where azurin/p28 stabilizing p53 by forming a binary complex is the most important and widely studied one. However, except the predictive structures given by molecular simulation and molecular docking, no information about the structure of p28-p53 or azurin-p53 complex is available. Consequently, it is impossible to rationally modify the amino acid sequence of p28 to further enhance the interaction between p28 and p53 and to improve the anticancer activity of p28. Through the comprehensive use of biophysical and structural biology characterization techniques and computer simulation, we systematically explored the interactions between p28 and the DNA binding domain (DBD) of p53. Through alanine mutagenesis, GST pull-down and surface plasmon resonance experiments, it was found that the L68A mutation had the greatest effect on the p28/p53 DBD binding affinity. Through fluorescence polarization experiments, the p28 binding site on p53 DBD was found to be overlapped with the DNA binding site. The p28-interacting residues on p53 DBD were further identified by nuclear magnetic resonance, including Y236, S240, V274, V173, R267, S215, I255, G245, L145, K305, M133, Y103, A161, R156, C277, and T211. Finally, the best binding model consistent with available experimental information was obtained through molecular docking.