Flocculation in Saccharomyces cerevisiae is a critical phenotype with ecological and industrial significance. This study aimed to functionally dissect the contributions of individual FLO genes (FLO1, FLO5, FLO9, FLO10, FLO11) to flocculation by employing an optogenetic circuit (OptoQ-AMP5) for precise, light-inducible control of gene expression. A FLO-null platform yeast strain was engineered allowing the expression of individual FLO genes without native background interference. Each FLO gene was reintroduced into the FLO-null background under the control of OptoQ-AMP5. Upon light induction, strains expressing FLO1, FLO5, or FLO10 demonstrated strong flocculation, with FLO1 and FLO5 forming large and structurally distinct aggregates. FLO9 induced a weaker phenotype. Sugar inhibition assays revealed distinct sensitivities among flocculins, notably FLO9’s novel sensitivity to fructose and maltotriose. Additionally, FLO-induced changes in cell surface hydrophobicity were quantified, revealing that FLO10 and FLO1 conferred the greatest hydrophobicity, correlating with their aggregation strength. This work establishes a robust platform for investigating flocculation mechanisms in yeast with temporal precision. It highlights the phenotypic diversity encoded within the FLO gene family and their differential responses to environmental cues. The optogenetic system provides a valuable tool for both fundamental studies and the rational engineering of yeast strains for industrial fermentation processes requiring controlled flocculation.