In order to study the effects of gallic acid (GA) on muscle quality and myoglobin conformation of bluefin tuna during refrigerated storage (4℃), different concentrations of GA (200mg/kg and 400mg/kg) were used to treat bluefin tuna and analyzed the changes in its quality characteristics. The effects of GA on the structure and microenvironment of myoglobin were further investigated by multispectral technique. The binding sites of the two proteins were determined by molecular docking. Results showed that during storage at 4℃, the quality characteristics of tuna meat exhibited varying degrees of deterioration, including a noticeable browning effect and a decrease in redness value. Compared with GA treatment group, lipid oxidation, metmyoglobin relative content, total volatile basic nitrogen and the total number of colonies in control group showed a significant upward trend. Additionally, there was a decrease in bound water content and an observed migration of water. Furthermore, varying degrees of muscle fiber bundle disruption were also noted. In conclusion, low concentration (200mg/kg) of GA could effectively alleviate the deterioration of quality of bluefin tuna. By multispectral analysis, low concentration of GA could stabilize the secondary structure of myoglobin well and had fluorescence quenching effect on myoglobin. Molecular docking results showed that myoglobin formed hydrogen bonds with GA through Leu 42, Pro 44 and His 42. This experiment provided basis and strategy for improving the quality of thawed bluefin tuna.