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 with the increase of H₂O₂ concentration, the carbonyl content of myosin increased by 2.376 times, while the contents of total sulfhydryl, free sulfhydryl, ionic bond and hydrogen bond decreased by 37.02%, 53.24%, 50.98% and 50.85%, respectively, upon treatment with 20 mmol/L H₂O₂ compared to the control group (0 mmol/L). 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, were 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 attacked 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.