Zeins were isolated from corn ethanol coproduct distiller’s dried grains (DDG) and fractionated into α- and β γ-rich fractions. The effects of the ethanol production process, such as fermentation type, protease addition, and DDG drying temperature on zein recovery, were evaluated. Yield, purity, and molecular properties of recovered zein fractions were determined and compared with zein isolated from corn gluten meal (CGM). Around 29−34% of the total zein was recovered from DDG, whereas 83% of total zein was recovered from CGM. Process variations of cooked and raw starch hydrolysis and fermentation did not affect the recovery, purity, and molecular profile of the isolated zeins; however, zein isolated from DDG of raw starch fermentation showed superior solubility and film forming characteristics to those from conventional 2-stage cooked fermentation DDG. Protease addition during fermentation also did not affect the zein yield or molecular profile. The high drying temperature of DDG decreased the purity of isolated zein. SDS-PAGE indicated that all the isolated α-zein fractions contained α-zein of high purity (92%) and trace amounts of β and γ-zeins cross-contamination. Circular dichroism (CD) spectra confirmed notable changes in the secondary structure of α-zeins of DDG produced from cooked and raw starch fermentation; however, all the α-zeins isolated from DDG and CGM showed a remarkably high order of α-helix structure. Compared to the α-zein of CGM, the α-zein of DDG showed lower recovery and purity but retained its solubility, structure, and film forming characteristics, indicating the potential of producing functional zein from a low-value coproduct for uses as industrial biobased product.