Abstract: Fermentation properties of 59 corn samples were determined by stimulating the dry mill process in laboratory, and the feasibility of using starch content and complete hydrolyzed starch (CHS) content as indicators to evaluate fermentation quality of corn for fuel ethanol production was explored. The microstructure of protein in mashed corn samples was observed by using a confocal laser scanning microscope (CLSM) to investigate the influence of protein cross-linking on fermentation conversion efficiency. The results showed that starch content of corn samples was positively correlated to ethanol yield significantly, but it only explained 81% of the ethanol yield variation. CLSM images displayed strong web-like protein matrix in mashed corn samples, and more compact microstructures were observed in samples with lower conversion efficiencies, indicating higher degree of protein cross-linking. The coefficient of determination between CHS content and ethanol yield was 0.909 6. It was more reliable for CHS content to predict ethanol yield than starch content, which was demonstrated by a validation test to a prediction model.