Nutrient fluctuation is ubiquitous in fermentation ecosystems. However, the microbial community assembly mechanism and metabolic characteristics in response to nutrient variation are still unclear. Here, we used Baijiu fermentation as a case example to study the responses of microbial community assembly and metabolic characteristics to the variation of amino acids using high-throughput sequencing and metatranscriptomics analyses. We chose two fermentation groups (group A with low amino acid and group B with high amino acid contents). The two groups showed similar succession patterns in the bacterial community, whereas they showed different succession in the fungal community wherein Pichia was dominant in group A and Zygosaccharomyces was dominant in group B. The β-nearest taxon index (βNTI) revealed that bacterial community was randomly formed, whereas fungal community assembly was a deterministic process. Variance partitioning analysis and redundancy analysis revealed that amino acids showed the largest contribution to the fungal community (37.64%, P = 0.005) and were more tightly associated with it in group B. Further study revealed that serine was positively related to Zygosaccharomyces and promoted its growth and ethanol production. Metatranscriptomic analysis revealed that the differential metabolic pathways between the two groups mainly included carbohydrate metabolism and amino acid metabolism, which explained the differences of ethanol production and volatile metabolites (such as isoamylol, isobutanol, and 2-methyl-1-butanol). Then these metabolic pathways were constructed and related gene expression and active microorganisms were listed. Our study provides a systematical understanding of the roles of amino acids in both ecological maintenance and flavor metabolism in fermentation ecosystems. IMPORTANCE In spontaneous fermented foods production, nutrient fluctuation is a critical factor affecting microbial community assembly and metabolic function. Revealing the microbial community assembly mechanism and how it regulates its metabolic characteristics in response to nutrient variation is helpful to the management of the fermentation process. This study provides a systematical understanding of the effect of amino acids on the microbial community assembly and flavor metabolisms using Baijiu fermentation as a case example. The data of this study highlight the importance of the nutrient management in fermentation ecosystems.