The solubility of zein varies with solvent types, therefore, affecting the physicochemical properties of film-forming solutions (FFS) and the fabricated zein films. The transformation of α-helix to β-sheet in zein molecules varied with solvent polarity, which influenced the self-assembly of zein. In this research, zein film was fabricated using various concentrations of aqueous acetic acid solvent (AAS) and aqueous ethanol solvent (AES). Zein was soluble over 96 % in 40–80 %(v/v) AAS and 60–80 % in 75–95 %(v/v) AES, respectively. SDS-PAGE reveals that the insolubility of the β-zein and dimers of α-zein was responsible for the solubility difference. The formation of the zein network affects the mechanical and physical properties of the film. The tensile strength of the film was over 25 MPa when the β-zein was soluble, and the zeta potential of FFS was over + 25 mV. Films fabricated with AAS had at least five times higher Young’s modulus than AES films. The transformation ratio of α-helix to β-sheet and reassembling of β-sheet into strips during self-assembly depended on the water percentage in the FFS. The free water stuck in the film network contributed to the moisture content of 8–10 % in AAS films and 11–16 % in AES films in films was observed by LF NMR. This study suggested that a stable FFS (zeta potential > +40 mV) and organized network in the film could be obtained by dissolving zein in AAS (≤ 70 %, v/v) than in AES.