The study aimed to investigate the effects of ultrasound treatment, varying in power and duration, on the solution behavior, multi-level structure, and functional properties of grape seed protein (GSP). Ultrasound significantly enhanced the functional properties of GSP, with optimal improvements at 400 W for 10 min. However, excessive treatment reduced these benefits. SDS-PAGE analysis confirmed the integrity of the primary protein structure after treatment. Circular dichroism spectroscopy revealed a reduction in ordered/stable protein secondary structures (alpha-helix and beta-sheet content, significantly decreased by 24.86% and 21.03% under the optimal ultrasound conditions, respectively, compared to untreated GSP), which was further corroborated by Fourier transform infrared spectroscopy. Intrinsic fluorescence spectroscopy demonstrated significant alterations in the tertiary structure of GSP. Ultrasound treatment improved free sulfhydryl content and surface hydrophobicity (significantly increased by 41.05% and 45.27%, respectively), thereby decreasing protein aggregation. Ultrasound treatment also reduced particle size and increased zeta potential (significantly changed by 54.97% and 24.58%, respectively), promoting protein unfolding and exposure of hydrophobic groups. These structural changes improved its solubility, gel strength, emulsifying and foaming properties, digestibility, and antioxidant activity. Notably, ultrasonication did not induce noticeable oxidative damage to the protein, as the carbonyl groups content increased by only approximately 20%. Collectively, these results demonstrate that properly controlled ultrasound treatment can significantly optimize the structure of GSP, thereby enhancing its functional performance and expanding its potential applications in the food industry.