Mixed fermentation with Saccharomyces cerevisiae and Pichia kudriavzevii has been shown to enhance wine aroma, yet the underlying mechanisms remain unclear. Monoculture of S. cerevisiae, monoculture of P. kudriavzevii, and mixed culture of S. cerevisiae and P. kudriavzevii were conducted, and the study analyzed and compared the biomass, flavor profile, and transcriptome responses of the three groups. Both yeast species exhibited growth inhibition in mixed culture, especially P. kudriavzevii. Significant differences were observed in three organic acids and the foremost 20 volatile compounds. Mixed fermentation enhanced esters (e.g., ethyl butyrate, isoamyl acetate) and volatile acids (e.g., hexanoic acid), but decreased isobutanol, phenylethyl alcohol, and quinic acid. Transcriptomic analysis revealed 294 and 332 differentially expressed genes (DEGs) in S. cerevisiae and P. kudriavzevii, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation results indicated that DEGs in mixed fermentation were concentrated in carbohydrate metabolism and amino acid metabolism. Our integrated analysis suggested that genes such as TDH2, TDH3, and ENO2 were pivotal for ester biosynthesis. Moreover, ADH1, ADH2, HPA3, ALD6, and ARO8 were associated with quinic acid synthesis. Furthermore, ILV2, ILV5, ALD6, and others were central to the production of isobutanol and phenylethyl alcohol.