Excessive levels of higher alcohols have negative impacts on the quality of Baijiu. Understanding the key microorganisms responsible for producing higher alcohols and their assembly processes is crucial for controlling higher alcohols in multi-species Baijiu fermentation. This study investigated the changes in volatile compounds, including higher alcohols, and microbial communities in the four fermentation cycles of Jiang-flavor Baijiu. Higher alcohols were the most abundant in the first cycle. Through metatranscriptomic and culture-dependent analysis, Saccharomyces cerevisiae and four non-Saccharomyces yeasts (Pichia kudriavzevii, Zygosaccharomyces bailii, Schizosaccharomyces pombe, and Torulaspora delbrueckii) were demonstrated to be the main contributors to higher alcohol (isobutanol, isoamylol, and 1-propanol) production, with S. cerevisiae producing the highest levels. A higher abundance of Saccharomyces resulted in the highest levels of higher alcohols in the first cycle. Moreover, our results revealed that yeast community was assembled from stochastic to deterministic processes with the increase of fermentation cycles, driven by lactic acid. These findings provide valuable insights into controlling higher alcohol production in Baijiu fermentation.