Oxidation is an important reason for the decline of quality of the aquatic products during processing and storage. In this study， we oxidized（0， 3， 6， 9 and 12 h） sea cucumber （Stichopus japonicus） myofibrillar proteins in different hydroxyl radical-generated system （0.01 mol/L FeCl3 / 0.1 mol/L VC / 10 mmol/L H2O2 and 0.01 mol/L FeCl3 / 0.1 mol/L VC / 50 mmol/L H2O2）. Then the indicators related to protein oxidation were evaluated. The aggregation of the protein was determined by electrophoresis and the microstructure of protein was observed using cold field scanning electron microscopy. The results indicated that when the concentration of H2O2 was 10 mmol/L and 50 mmol/L， the carbonyl content reached （4.31±0.32） nmol/mg protein and （3.72±0.30） nmol/mg protein at 3 h and 12 h. The content of free-SH decreased from 0 h （12.17 ± 0.54） μmol/g protein to 12 h （7.27 ± 0.52） μmol/g protein and （6.64 ± 0.17） μmol/g protein. The fluorescence content showed a decreasing trend with the increase of oxidation time and reached the lowest value at 12 h. Under the treatment conditions of 50 mmol/L H2O2， the free amino group content decreased significantly with the increase of oxidation time， and decreased to 65% of the control group content at 12 h. Electrophoretic analysis showed that under non-reducing conditions， the degree of protein aggregation increased with the prolongation of oxidation time， and the degree of protein aggregation decreased after the addition of β-mercaptoethanol. The microstructure indicated that as the oxidation time increased， the myofibrillar protein had a compact structure and the degree of hinge of the protein was higher. In summary， the structural changes of the sea cucumber myofibrillar protein caused by oxidation in the hydroxyl radical system. This research has certain theoretical significance for controlling the quality of participation in the sea cucumber.