alpha-Zein has received widespread attention owing to its unique solubility, amphipathic, and self-assembly properties, which is because of its high proportion of nonpolar amino acids and unique amino acid sequence. The protein self-assembly is a significant and widely observed phenomenon in many scientific areas such as food and biomedicine, among many industries. In this study, we investigated the self-assembly behavior of alpha-zein and regulated the morphology and structure of the self-assembled alpha-zein by varying the experimental parameters like pH, ethanol content, induction time, and alpha-zein concentration during the self-assembly process in ethanol-water mixtures. The nanospheres and nanofibers were observed under different conditions [nanospheres observed under acidic and strongly alkaline (pH > 10.5) conditions or for ethanol content lower than 65% and higher than 75%; nanofibers observed under weakly alkaline (pH 9.5-10.5) conditions or for 65-75% ethanol concentration for induction duration longer than 24 h]. The morphological and structural analyses of the self-assembled alpha-zein showed that the self-assembly process was accompanied by the transformation of the morphology and conformation of alpha-zein. The studies on the self-assembly process and mechanism revealed that alpha-zein first self-assembled into nanospheres, followed by the nanospheres adhering to shape-beaded fibers and finally fibers, accompanied by a structural transformation from the disordered into ordered state. The nanosphere formation is noted to follow the nucleation-based polymerization, and the nanosphere-mediated mechanisms lead to the formation of nanofibers. Moreover, the hydrophobic interactions, hydrogen bonds, and electrostatic interactions are concluded to drive the alpha-zein self-assembly. The findings from this study are expected to provide a theoretical basis for expanding the commercial applications of alpha-zein.