Quercetin is a plant flavonol from the flavonoid group of polyphenols; it has medical importance in treating many diseases. Quercetin flavonoid was assessed using Rhodamine B Isothiocyanate (RBITC) dye in absence and presence of SiO₂ nanoparticles at pH= 7 using Tris-HCl buffer solution. This method considered a simple, sensitive, and selective method where the dye has fluorescence emission band at 583 nm after excitation at 552 nm which strongly quenched by quercetin. The results showed that presence of SiO₂ nanoparticles which were characterized using different techniques such as XRD, SEM, EDX, IR, TGA and TEM analysis has a significant effect on lowering the detection limit for quercetin using RBITC dye from 19.99 to 9.86 n mol.L⁻¹, increasing the binding constant from 7.17 × 10³ to 25.92 × 10³ mol⁻¹.L in absence and presence of SiO₂ nanoparticles respectively. The interaction between quercetin and RBITC dye was of hydrophobic type and the fluorescence quenching mechanism was static in absence and presence of SiO2 nanoparticles respectively. The method was applied for the determination of quercetin in spiked human plasma where recovery measurements proved the accuracy of this method.