A combination of genotype, cultivation environment, and protein separation procedure was used to modify the nanoscale morphology, polymerization, and chemical structure of glutenin proteins from wheat. A low-polymerized glutenin starting material was the key to protein-protein interactions mainly via SS cross-links during film formation, resulting in extended β-sheet structures and propensity toward the formation of nanoscale morphologies at molecular level. The properties of glutenin bioplastic films were enhanced by the selection of a genotype with a high number of cysteine residues in its chemical structure and cultivation environment with a short grain maturation period, both contributing positively to gluten strength. Thus, a combination of factors affected the structure of glutenins in bioplastic films by forming crystalline β-sheets and propensity toward the ordered nanostructures, thereby resulting in functional properties with high strength, stiffness, and extensibility.