Abstract: This study aimed to elucidate the interactions among β-conglycinin (7S), glycinin (11S), and glycerol, as well as their effects on film-forming properties. Molecular dynamics simulations with experimental characterization were systematically combined to analyze changes in molecular interactions and their impact on the physicochemical properties of protein films. The results showed that as the glycerol content increased, the radius of gyration and solvent-accessible surface area between the 7S and 11S globulins gradually increased, while the number of hydrogen bonds, electrostatic interactions, van der Waals forces, non-polar solvation energies, and absolute value of binding free energies progressively decreased. At a glycerol concentration of 40%, interactions such as hydrogen bonding and van der Waals forces between glycerol and the 7S-11S complex reached a maximum, and glycerol began to exhibit aggregation. As glycerol content increased from 10% to 50% in the 7S-11S protein films, the elongation at break (EAB) increased from 13.72% to 208.01%, while tensile strength (TS) decreased from 30.62 MPa to 2.51 MPa. A strong correlation was observed between TS, EAB, and the absolute value of the binding free energies, with a correlation coefficient exceeding 0.8. Glycerol addition enhanced the film peak intensity at 1040 cm⁻¹ in the Fourier-transform infrared spectrum. The proportion of hydrogen bonds and hydrophobic interactions between proteins in the 7S-11S films decreased with increasing glycerol content. Additionally, the high molecular weight components involved in hydrophobic interactions gradually decreased. The water contact angles on both film surfaces decreased from 115.82° and 112.33° to 94.00° and 84.33°, respectively, as the glycerol content increased. These findings suggest that glycerol modulates the intermolecular forces between 7S and 11S globulins, shifting the hydrophobic interactions among high-molecular-weight components toward ionic and hydrogen bonds, thereby affecting the mechanical properties and surface characteristics of the films.