This research systematically explored the influences of different ultrasonic treatment (UT) conditions (power: 0-800 W; time: 0-20 min) on the structure and gel characteristics of myosin obtained from Hypomesus olidus. Experimentally, UT markedly changed the aggregation behavior and molecular conformation of myosin. Under optimal conditions (200 W, 10 min), the particle size of myosin decreased by 62.05%, solubility increased by 3.19-fold, surface hydrophobicity increased markedly, and both total and free sulfhydryl contents decreased. FTIR analysis showed that UT decreased the α-helix and antiparallel β-sheet contents while increasing the β-sheet and random coil proportions, suggesting partial unfolding of the myosin composition. Fluorescence and UV spectroscopy further confirmed tertiary structure disruption while raising the exposure of aromatic amino acid residues. Regarding gel properties, appropriate UT (200 W, 10 min) improved gel whiteness (by 3.81%) and facilitated the creation of a more compact and uniform gel network, while improving viscoelasticity (G′ and G″). Excessive ultrasound power (≥400 W) or prolonged treatment time (≥15 min) resulted in protein re-aggregation or structural loosening. In conclusion, moderate UT effectively modulates the structure and functionality of Hypomesus olidus myosin, laying a groundwork for its high-value application in surimi processing.