A detailed photophysical characterization of biologically imperative drugs, 3-hydroxyflavone (3HF) and quercetin (QCT) upon binding with taurula yeast RNA (ribonucleic acid) has been studied. The steady-state absorption, emission and anisotropy profiles of these anti-cancer drugs evidence for the binding interaction through interconversion between prototropic conformations. Although diminution of fluorescence (at ∼ 515 nm for 3HF and ∼ 545 nm for QCT) was attributable to the protonated form indicating suppression of ESIPT phenomenon, the excitation spectra aids in identification of the actual conformation of the drugs after prototropic conversion. The dynamical aspects were displayed by time-resolved fluorescence decay and rotational relaxation behavior (‘dip-and-rise’ decay pattern) of the drugs within RNA helix. The quenching study hints for the probable binding location and the mode of binding but the circular dichroism study confirms it to be groove binding. This study endows with not only the biological insight of RNA-binding of naturally abundant flavonols but also photophysical depiction of spectroscopically challenging phenomenon in an aspect of its structural interconversion. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim