The dissolution, aggregation, and structural properties of zein in different solvents (methanol, ethanol, isopropanol, acetic acid, and acetone) were studied using ultraviolet spectroscopy, Fourier-transform infrared spectroscopy, fluorescence spectroscopy, and scanning electron microscopy. The surface hydrophobicity of the zein protein film was determined by static contact angle measurements. Zein showed good dissolution in 100% acetic acid, 80% ethanol, and isopropanol solutions, and the resulting solutions exhibited high light transmittance. A maximum particle size of 5230.16 nm and a minimum particle size of 25.52 nm were obtained when zein was dissolved in 70% acetic acid and 80% ethanol solutions, respectively. Zein dissolved in isopropanol showed high fluorescence absorption. Zein dissolved in 80% acetic acid and ethanol solutions showed contact angles of 88.90° and 86.60°, respectively, with the containers, suggesting neutral wettability and suitability of the solutions for stabilizing oil-water interfaces. The air-side contact angle of the zein film formed in 80% methanol solution was the largest (72.10°). These results revealed that the solvent concentrations affected the water-protein interactions, thereby resulting in different aggregation degrees of the proteins. In addition, differences in the solvent polarity and electronegativity also affected the particle size, surface hydrophobicity, and spatial conformation of the protein by changing the hydrophobic interactions between the molecules and formation of hydrogen and disulfide bonds.