The goal of this study was to develop conditional flocculant yeasts for use in the alcohol fermentation industry. Promoters were constructed to completely repress gene transcription in the presence of sugar and to support strong expression after the exhaustion of this compound. A fusion of regulatory regions of the ADH2 promoter with the FLO5 core promoter was constructed to regulate the FLO5 gene. This construct was inserted into multicopy plasmids and transformed into laboratory strains of Saccharomyces cerevisiae, whereby the transformed cells were selected by sedimentation from the bulk medium after sugar exhaustion, without decreasing ethanol production. The ADH2-FLO5 region was converted into an integrative cassette to disrupt the CAN1 gene in industrial yeast strains. Transformed cells became resistant to canavanine and demonstrated conditional flocculation. Although ethanol production was significantly decreased in the industrial transformants, this development reveals a promising technology for the substitution of centrifugation in industrial ethanol production.