(Figure Presented) Ruthenium(II) complexes have rich photophysical attributes, which enable novel design of responsive luminescence probes to selectively quantify biochemical analytes. In this work, we developed a systematic series of Ru(II)- bipyrindine complex derivatives, [Ru(bpy) 3-n(DNP-bpy) n](PF 6) 2 (n = 1, 2, 3; bpy, 2,2′-bipyridine; DNP-bpy, 4-(4-(2,4- dinitrophenoxy)phenyl)-2, 2′-bipyridine), as luminescent probes for highly selective and sensitive detection of thiophenol in aqueous solutions. The specific reaction between the probes and thiophenol triggers the cleavage of the electron acceptor group, 2,4-dinitrophenyl, eliminating the photoinduced electron transfer (PET) process, so that the luminescence of on-state complexes, [Ru(bpy) 3-n(HP-bpy) n] 2+ (n = 1, 2, 3; HP-bpy, -(4-hydroxyphenyl)-2,2′- bipyridine), is turned on. We found that the complex [Ru(bpy)(DNP-bpy) 2] 2+ remarkably enhanced the on-to-off contrast ratio compared to the other two (37.8 compared to 21 and 18.7). This reveals a new strategy to obtain the best Ru(II) complex luminescence probe via the most asymmetric structure. Moreover, we demonstrated the practical utility of the complex as a cell-membrane permeable probe for quantitative luminescence imaging of the dynamic intracellular process of thiophenol in living cells. The results suggest that the new probe could be a very useful tool for luminescence imaging analysis of the toxic thiophenol in intact cells.