A pair of chiral ruthenium(II) complexes, Lambda- and Delta-[Ru(bpy)(2)(p-BEPIP)] (ClO4)(2) [Lambda- and Delta-RM0627; bpy = 2,2-bipyridine; p-BEPIP = 2-(4-phenyacetylenepheny1)-1H-irnidazo[4,5f] [1,10]phenanthroline], were prepared using the Sonogashira coupling reaction under microwave irradiation. The study shows that Lambda-RM0627 emitted strong phosphorescence in the range 500-700 nm with a maximum at 594 nm when excited at 365 nm (the Stokes shift is about 227 nm), which was mainly located in the cell nucleus with red phosphorescence. Further studies using real-time phosphorescence observation confirmed that Lambda-RM0627 can be taken up quickly by MDA-MB-231 cells and enriched in the nucleus. The in vitro and in vivo toxicities of Lambda-RM0627 were also evaluated, and it was found that Lambda-RM0627 slightly inhibited the growth of MDA-MB-231 breast cancer cells and HaCaT normal human epidermal cells and had little influence on the development of Zebrafish embryos at low concentration. In conclusion, the levoisomer of chiral ruthenium complexes can act as a potential phosphorescent probe that targets nuclei of living cells with low toxicity.