A unique ruthenium(II) complex, tris(4-methyl-2,2′-bipyridyl- 4′-carboxaldehyde)Ru(II) hexafluorophosphate [Ru(CHO-bpy) 3](PF6)2, has been designed and synthesized as a highly sensitive and selective luminescence probe for the recognition and detection of cysteine (Cys) and homocysteine (Hcy). The almost non-luminescent probe can rapidly react with Cys and Hcy to yield the corresponding thiazolidine and thiazinane derivatives, accompanied by the remarkable luminescence enhancement and a large blue-shift of the maximum emission wavelength from 720 to 635 nm. The dose-dependent luminescence enhancement of the probe shows a good linearity in the Cys/Hcy concentration range of 15 to 180 μM with the detection limits of 1.41 μM and 1.19 μM for Cys and Hcy, respectively. Furthermore, the luminescence response of the probe is highly specific to Cys/Hcy only even in the presence of various amino acids, protein, and DNA. The results of this work not only demonstrate the efficacy and advantages of the Ru(II) complex-based luminescence probe for the sensitive and selective detection of Cys/Hcy but also provide a useful strategy for the rational design of Ru(II) complex-based luminescence probes for various biological molecules.