Rhodamine B selenolactone has been designed, synthesized, and characterized as a new fluorescent probe for imaging both Hg2+ and Ag+ in live cells to better understand their distinct toxicities to organisms. The probe is designed based on the fact that selenium has a strong affinity for mercury and silver, and is constructed by Incorporating a Se atom into the spirocyclic structure of rhodamine. It exhibits a rapid and specific spectroscopic off-on response to Hg2+ and Ag` instead of other species, with detection limits of 23 nM Hg2+ and 52 nM Ag+. Moreover, the probe is membrane-permeable, and can react with Ag+ even in the presence of Cl- because of the higher affinity of Se than Cl- for Ag+, which makes it of potential use for imaging not only Hg2+ but also Ag+ in live cells. This applicability has been demonstrated by imaging Hg2+ and Ag+ in Hela cells. It is observed that the reaction of Ag+ with the probe insido the cells occurs much slower than that of Hg2+, which is ascribed to the high concentration of cellular chloride ions inhibiting the formation of sufficient free Ag+. The present finding is helpful to get an insight into the different interaction mechanism of Hg2+ and Ag+ with cells, and more applications of the probe may be expected for studying the behaviors of Hg2+ and Ag+ in various biosystems.