Abstract: In this study, myosin was extracted from fresh mutton using precipitation centrifugation, purified via column chromatography, and concentrated through ammonium sulfate salting-out combined with ultrafiltration centrifugation. The results demonstrated that the improved protein extraction method was more efficient. The optimal loading amount of myosin onto the ion-exchange column was determined to be 20 mg, and the optimal saturation level for ammonium sulfate salting-out stood at 50%. To explore the impacts of hydroxyl radical oxidation on the structure and oxidation sites of myosin, a hydroxyl radical oxidation system was established by regulating the concentration of H₂O₂ (0, 0.5, 1, 5, 10, and 20 mmol/L), which enabled myosin to undergo oxidation to varying degrees. Research findings indicated that as the H₂O₂ concentration increased, the carbonyl content of myosin was 2.376 times higher than that of the control group. Meanwhile, the total sulfhydryl content decreases by 37.02%, the free sulfhydryl content by 53.24%, the ionic bond content by 50.98%, and the hydrogen bond content by 50.85%. In contrast, the disulfide bond content exhibited a tendency of first increasing and then decreasing. It reached its maximum value when the H₂O₂ concentration was 10 mmol/L, which was 2.28 times that of the control group. Measurements using a fully automatic amino acid analyzer revealed that four amino acids, namely Cys, Tyr, Phe, and His, are susceptible to oxidation, with the order of their susceptibility being Cys>Tyr>His>Phe. LC-MS/MS detection showed that under mild oxidation conditions, hydroxyl radicals mainly attack the proteins around the SH1-SH2 region. When subjected to severe oxidation at an H₂O₂ concentration of 20 mmol/L, a large amount of myosin aggregated, and these aggregations primarily occurred in the head helical region and the 1502-1783 region of myosin.