Abstract: To optimize the preparation process of peanut peptide-selenium complex assembly and explore the changes in its structural characteristics. In this paper, firstly, the selenium binding rate of peanut peptide was used as the main indicator, and the protein hydrolysis degree as the auxiliary indicator to screen out the peanut peptide fragments obtained by biological enzymatic hydrolysis technology. Then, the preparation process of peanut peptide-selenium complex was optimized through single-factor and response surface experiments, and the changes in the structure of peanut peptide after combination with selenium were studied by modern spectroscopic techniques such as ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and fluorescence spectroscopy. The results showed that the selenium binding rate of the peanut protein hydrolysate was the highest, when the alkaline protease hydrolyzed peanut protein for 2.5 h. The single-factor test showed that the effect of the mass ratio of peanut peptide to selenium, temperature, and pH on the preparation process of peanut peptide-selenium complex was greater than that of reaction time. Therefore, three factors of the mass ratio of peanut peptide-selenium, temperature, and pH were selected to optimize the preparation process of the peanut peptide-selenium complex in the response surface test. The optimal preparation process was finally obtained as the mass ratio of peanut peptide to selenium was 4∶1, the pH was 9.0, the temperature was 83 ℃, and the reaction time was 60 min. The selenium binding rate of the peanut peptide-selenium complex was 75.17%. The spectroscopic results showed that the spatial structure of the peanut peptide changed after the complex reaction, indicating that the amino, carboxyl and carbonyl groups in the peanut peptide were involved in the complex reaction. In this study, the optimum conditions for the preparation of peanut peptide-selenium complex were identified and the sites involved in the selenium complex reaction of peanut peptide were preliminaries explored, providing an important reference for the development of novel peptide selenium supplements.