Abstract: Stacking fermentation is a crucial step in producing sauce-flavor Baijiu. The relationship between microbial diversity and volatile flavor substances is complex and dynamic. To better understand the correlation between microorganisms and flavor substances in Dongniang sauce-flavor Baijiu during the five-round stacking fermentation of Jiupei, high-throughput sequencing (HTS) technology and headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) were used to identify the microbial flora diversity and volatile flavor compounds. In addition, the Pearson correlation coefficient was computed, and a visualizable correlation network diagram was drawn based on the detection results. The results identified 55 volatile flavor compounds, including 25 esters, 5 alcohols, 7 aldehydes, 4 acids, 4 phenols, 3 alkanes, and 7 others. The main volatile components were ethyl lactate, phenylethyl alcohol, ethyl phenylacetate, and phenethyl acetate. The dominant microorganism genera in the floral structure were Virgibacillus, Kroppenstedtia, Bacillus, Oceanobaciillus, Thermomyces, Zygosaccharomyces, and Thermoascus. Kroppenstedtia, Monascus, Bacillus, and Zygosaccharomyces significantly affected flavor substances among the flora. Overall, the purpose and significance of this study are to provide a theoretical foundation for enhancing the quality of Dongniang sauce-flavor Baijiu.