<P><B>Abstract</B></P> <P>The aim of this study was to construct a cost-effective method for repeated bioethanol production using membrane (ultrafiltration permeation and nanofiltration concentration)-concentrated sweet sorghum juice by using flocculent <I>Saccharomyces cerevisiae</I> F118 strain. With low initial dry cell concentrations at around 0.28–0.35 g L<SUP>−1</SUP>, the <I>S. cerevisiae</I> F118 strain provided an ethanol titer of 86.19 ± 1.15 g L<SUP>−1</SUP> (theoretical ethanol yield of 70.77%), which was higher than the non-flocculent <I>S. cerevisiae</I> BY4741 strain at 33.92 ± 0.99 g L<SUP>−1</SUP> after 24 h fermentation. This result was correlated with higher gene expressions of the sucrose-hydrolysing enzyme invertase, sugar phosphorylation, and pyruvate-to-ethanol pathways in the F118 strain compared with the BY4741 strain. Sequential fed-batch fermentation was conducted, and the F118 strain was easily separated from the fermentation broth via the formation of flocs and sediment. After the 5th cycle of fermentation with the F118 strain, the ethanol concentration reached 100.37 g L<SUP>−1</SUP>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Flocculent yeast F118 strain was easily separated from the fermentation broth. </LI> <LI> The membrane-concentrated sweet sorghum juice was used as a feedstock. </LI> <LI> Fermentation could be started with low cell amounts (0.28–0.35 g-dry cells L<SUP>−1</SUP>). </LI> <LI> Expression of genes related to invertase and pyruvate-to-ethanol pathways was high. </LI> <LI> F118 strain showed high ethanol productivity at repeated fed-batch fermentation. </LI> </UL> </P>