In this work, we designed and synthesized a heterobimetallic ruthenium(ii)-nickel(ii) complex, [Ru(bpy)2(phen-DPA)Ni](PF6)4 (Ru-Ni), as a highly selective phosphorescence probe for histidine. The probe exhibited weak emission at 603 nm because the phosphorescence of the Ru(ii) complex can be strongly quenched by the paramagnetic Ni(2+) ion. In the presence of histidine, reaction of Ru-Ni with histidine resulted in the release of nickel(ii) and an enhancement in the phosphorescence intensity at 603 nm. Ru-Ni showed high selectivity for histidine even in the presence of other amino acids and cellular abundant species. Cell imaging experimental results demonstrated that Ru-Ni is membrane permeable, and can be applied for visualizing histidine in live cells. More interestingly, Ru-Ni also can act as a novel reaction-based nuclear staining agent for visualizing exclusively the nuclei of living cells with a significant phosphorescence enhancement. In addition, the potential of the probe for biological applications was confirmed by employing it for phosphorescence imaging of histidine in larval zebrafish and Daphnia magna. These results demonstrated that Ru-Ni would be a useful tool for physiological and pathological studies involving histidine.