The role of Mg2+ ion in flavin (flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)) recognition by RNA aptamer has been explored through steady state and time-resolved fluorescence, circular dichroism (CD), thermal melting (TM) and isothermal titration calorimetry (ITC) studies. A strong quenching of flavin emission is detected due to stacking interaction with the nucleobases in the mismatched region of aptamer, and it enhances manifold with increasing Mg2+ concentrations. A comparatively lower binding affinity toward FAD compared to FMN is attributed to the presence of intramolecular 'stack' conformer of FAD, which cannot participate in the intermolecular stacking interactions with the nucleobases. CD and TM studies predict that flavin detection causes structural reformation of RNA aptamer. ITC results indicate that flavin detection is thermodynamically feasible and highly enthalpy driven. © 2014 Published by Elsevier B.V.