Abstract: In this study, myosin isolated from Nemipterus virgatus was added with 0%~8%（w/w）soybean protein isolate (SPI) and the physicochemical, spectroscopic, and gel properties of the composite system were analyzed, in order to investigate the effect of interaction between myosin and SPI on the gel properties of myosin. The results showed that after the addition of SPI, the endogenous fluorescence intensity of the composite system decreased, whereas the ultraviolet absorbance, the total sulfhydryl content, and hydrogen bond as well as hydrophobic interactions increased. These results indicated that there were the hydrophobic interactions, hydrogen bonds, disulfide bonds, and other covalent and non-covalent interactions between SPI and Nemipterus virgatus myosin, which altered the structure and conformation of myosin. Meanwhile, the gel strength, hardness, and β-sheet content of the myosin-SPI composite gels all increased initially and then decreased as the increase addition of SPI. Furthermore, the total sulfhydryl and hydrophobic contents of the myosin-SPI composite system got the highest values when added 4% SPI, which increased by 64.46% and 13.53% compared with the control group, respectively. The gel strength and hardness of the myosin-SPI composite gels were also reached the highest values when added 4% SPI, which increased by 8.66% and 9.21% compared with the control group, respectively. It was also found that a part of α-helices transformed into β-sheets and the gel network was most compact and homogeneous. Therefore, the moderate addition of SPI could promote the aggregation and cross-linking of myosin through the interaction forces such as hydrophobic interactions, hydrogen bonds, and disulfide bonds, and improve the gel properties of myosin.