Abstract: In this study, bovine blood was utilized as raw material, the extracted bovine hemoglobin was enzymolized, and then the most suitable enzymolysis protease was screened. Single factor combined response surface experiments were used to explore the optimal enzymolysis process. The structure of the prepared bovine hemoglobin peptide iron chelate (BHP-Fe) was characterized by technologies, such as scanning ultraviolet spectrum, Fourier infrared spectrum, scanning electron microscopy, and fully automated amino acid analyzer. Its stability in vitro was investigated through thermogravimetric analysis and simulated digestion of gastrointestinal tract in vitro. The results showed that the stepwise enzymatic hydrolysis of pepsin and alkaline protease was the most suitable enzymatic solution. Based on the preliminary enzymolysis of pepsin, optimal conditions for alkaline protease enzymolysis were obtained: A solid-liquid ratio of 1:3, an enzymolysis pH of 9.8, an enzymolysis temperature of 41 ℃, an enzyme dosage of 5900 U/g, and an enzymolysis time of 2 h. Under these conditions, the Fe²⁺ chelating capacity of bovine hemoglobin peptide (BHP) obtained through enzymatic hydrolysis was 72.11%, and the degree of bovine hemoglobin hydrolysis was 35.07%. The results of ultraviolet and Fourier infrared spectra showed that Fe²⁺ chelated with carboxyl oxygen and amino nitrogen on the BHP peptide chain, producing new substances different from polypeptides. Scanning electron microscopy revealed that the peptides changed from smooth fragments to rough patches after chelating Fe²⁺, and there were obvious differences between them. The change in amino acid content indicated that aspartate, glutamic acid, and lysine serve as binding sites for polypeptides and metal ions. Thermogravimetric analysis results demonstrated that BHP and BHP-Fe remain stable at high temperatures, maintaining good stability even at 300 ℃. The results of in vitro simulated digestive stability analysis showed that BHP-Fe could maintain higher stability in the gastrointestinal tract than other iron supplements. In conclusion, the polypeptide-derived iron supplement prepared in this experiment had strong chelating force, good digestive stability, and broad application prospects, which could provide constructive reference for the efficient utilization of bovine blood by-products and the future development prospect of new polypeptide-derived iron supplement.