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Abstract The interaction of [Ru(phen) 2 dppz-idzo]2+ (phen = 1,10-phenanthroline, dppz-idzo = dppz-imidazolone) with triplex RNA poly(U)·poly(A)*poly(U) was carried out by using spectroscopic and viscometric techniques in this work. Luminescent titrations suggest that [Ru(phen) 2 dppz-idzo]2+ shows better selectivity for poly(U)·poly(A)*poly(U) compared with poly(U)·poly(A) and poly(U), this complex exhibits a "light switch" effect with an emission enhancement factor of about 123 in the presence of poly(U)·poly(A)*poly(U). Significantly, this "light switch" behavior could even be observed by the naked eye under irradiation with UV light. To our knowledge, [Ru(bpy) 2 dppz-idzo]2+ is the first small molecule able to serve as a colorimetric molecular "light switch" for the triplex poly(U)·poly(A)*poly(U). Combined with the spectral and viscometric results as well as [Ru(phen) 2 dppz-idzo]2+ stabilizing the template duplex poly(U)·poly(A), we speculate that [Ru(phen) 2 dppz-idzo]2+ prefers to bind with the Hoogsteen base-paired strand (the third strand) of the triplex, thus the intercalating [Ru(phen) 2 dppz-idzo]2+ stabilizing the third strand is more marked in comparison with the Watson-Crick base-paired duplex of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)·poly(A)*poly(U) with small molecule, particularly ruthenium(II) complexes. Graphical Abstract The present study reveals that [Ru(phen) 2 dppz-idzo]2+ exhibits stronger binding with the Hoogsteen base-paired strand than to the Watson-Crick base-paired duplex of the RAN triplex poly(U)·poly(A)*poly(U), thereby stabilizing the Hoogsteen base-paired strand more effectively under the same conditions. Viscometric experiments reveals that the binding mode of [Ru(phen) 2 dppz-idzo]2+ toward the RNA triple is intercalation. The fluorescence titrations indicate that [Ru(phen) 2 dppz-idzo]2+ is able to serve as a colorimetric molecular "light switch" for the RNA triplex. CD titrations indicate that [Ru(phen) 2 dppz-idzo]2+ can induce significant conformational perturbations of the RNA triplex. The present results further offer valuable information on the interaction of the RNA triplex with small molecules, particularly ruthenium(II) complexes. Unlabelled Image Highlights • [Ru(phen) 2 dppz-idzo]2+ exhibited a "light switch" effect for the RNA triplex. • This "light switch" behavior could even be observed by the naked eye. • [Ru(bpy) 2 dppz-idzo]2+ is a colorimetric molecular "light switch" for the RNA triplex. • [Ru(phen) 2 dppz-idzo]2+ stabilizes the third strand was more marked. [ABSTRACT FROM AUTHOR]