<p>Flavonoids are naturally occurring antioxidants with a variety of biological activity. Fluorescence techniques can be used to investigate their molecular interactions. Using a combination of computational and experimental methods, we examined the photoinduced interaction between a number of structurally different flavonoids and the cationic fluorescent dye Nile Blue (NB) in this work. Flavonoid-induced dampening of NB emission and ground-state complex formation were validated by steady-state fluorescence studies and UV-visible absorption. The efficiency of the quenching was better in polar protic media and under basic circumstances, and it was reliant on both pH and solvent. Studies of time-resolved fluorescence revealed a static quenching mechanism, which was corroborated by spectrum shifts and thermodynamic characteristics. Bimolecular quenching rate constants (k_q) were correlated with bond dissociation enthalpy (BDE), hydroxyl location, and flavonoid structure. Strong predicted correlations between quenching behavior and flavonoid electronic characteristics were also found using partial least squares (PLS) regression. The antioxidant activity trend was confirmed by deoxyribose degradation assays, which were in line with quenching efficiency. The way flavonoid structural characteristics control NB quenching and antioxidant ability is highlighted by this integrated investigation. The findings of this study provide a foundation for developing advanced antioxidant screening tools, fluorescence-guided therapeutic platforms, and flavonoid-based biosensors for biomedical and environmental applications.</p>