To enhance the physicochemical properties of zein nanoparticles, zein complexes with two Gemini surfactants (12-3-12 and 12-4-12) were prepared using the anti-solvent method and investigated the physicochemical properties, formation mechanism and antibacterial activity. Results indicated that the optimal mass ratio between zein and Gemini surfactants was at 1:1, and the incorporation of Gemini surfactants significantly improved the surface properties of zein, reducing its surface hydrophobicity and surface tension, thereby enhancing its dispersion in aqueous media. Fluorescence spectroscopy and molecular docking experiments further elucidated the interaction mechanisms between zein and Gemini surfactant, revealing a spontaneous binding process, mainly driven by hydrophobic and hydrogen interaction, and a strong binding affinity of 12-4-12 with zein. Additionally, the zein/Gemini surfactant complexes exhibited significant antibacterial activity against Staphylococcus aureus, with the zein/12-4-12 complex showing particularly prominent inhibitory effects. Therefore, this research not only provides a theoretical foundation for the construction of Gemini surfactant stabilized zein nanoparticles but also points the way for the subsequent embedding of bacteriostatic agents to achieve synergistic antibacterial effects.