High-throughput sequencing was used to define microbial community structure and GC-MS to identify volatile flavor substances during fermentation of corn yellow wine, and results were analyzed by multivariate statistical analysis. Seventeen bacterial phyla, 239 bacterial genera, 4 fungal phyla, and 18 fungal genera were found and changes in community structure occurred during fermentation. Twenty-four volatile flavor substances, including 14 esters and 5 alcohols, were detected and changes during fermentation recorded. Sixteen microbial genera correlated with volatile flavor substances and Weissella, Lactobacillus, Pseudomonas, Rhodotorul, and Kwoniella had significant correlation with ethyl esters and higher alcohols. Micro-organisms thus influence flavor development during corn yellow wine fermentation.