This study mainly evaluated the effect of different energies of pulsed light (PL) treatment (100, 200, 300, 400, and 500 J/pulse) on myofibrillar protein (MP) of large yellow croaker during refrigerated storage. The results showed that PL treatment would cause a certain degree of oxidation to the MP of large yellow croaker at the initial stage, which showed that the total sulfhydryl content of the protein decreased, the carbonyl content and the average particle size increased, and the β‐sheet to β‐turn transformation, the tertiary structure of the protein unfolds, and the hydrophobic groups were exposed, causing the reduction of intrinsic fluorescence intensity. However, subsequent storage studies found that PL treatment could slow down the oxidation rate of MP. The decrease rate of total sulfhydryl content and the increase rate of carbonyl content in the 300 J/pulse group were both reduced by about 1.7 times compared with the control group. At the same time, the PL treatment with this intensity could also better protect the secondary structure, tertiary structure, and microstructure of MP. This study provided theoretical basis and reference for analyzing the quality change rule and mechanism of large yellow croaker during refrigerated storage after PL treatment. Studies have shown that PL treatment can reduce the adverse changes of MP in large yellow croaker during cold storage.