Phenolic acid-chitosan derivatives have received extensive attention due to their greatly enhanced mechanical, antibacterial and antioxidant properties, especially in food preservation. The chitosan-gentianic acid (CS-g-GA) was prepared and its impact on myofibrillar proteins (MPs) in sea bass (Lateolabrax maculatus) during refrigerated storage was investigated in this study. Fish fillets were immersed in distilled water, CS, GA and CS-g-GA solutions, respectively, followed by an 18-day refrigerated storage. MPs were extracted from fish fillets every 3 days, and then the degree of oxidation and conformational changes of MPs were evaluated. The results of myofibril fragmentation index (MFI) and surface hydrophobicity confirmed that CS-g-GA can retard the fragmentation and degradation of myofibrils. Additionally, the total sulfhydryl content of CS-g-GA-treated samples in the later storage was significantly higher than those of other groups, confirming that CS-g-GA treatment protected the structure and function of protein. Results of Fourier transform infrared spectroscopy (FT-IR) and inner fluorescence intensity (IFI) showed that CS-g-GA maintained the secondary and tertiary structure of MPs. The results from scanning electron microscope (SEM) further clarified that CS-g-GA can maintain the tight arrangement of muscle fibers and protect the microstructure of myofibrils. Furthermore, the observation of confocal laser scanning microscopy (CLSM) demonstrated that CS-g-GA can prevent protein cross-linking and aggregation. Therefore, CS-g-GA treatment can effectively protect the structure and delay the oxidation of MPs.