A new class of fluorescein/rhodamine hybrids with two spirolactone rings was reported to exhibit dual-output fluorescent behaviors independently. Isolation and characterization for two diastereomers, trans-RhOH and cis-RhOH, have been made and their X-ray crystal structures determined. In a basic environment, the spirolactone ring on the hydroxyl side will be opened to give a fluorescein-like optical output with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm absorption and 575 nm emission, will be switched on by a H+ or a Hg2+ ion, attributed to the spirolactone ring opening on the amino side. In a methanol-buffer system with different pH values, the corresponding pK(a) values for the hydroxyl and amino groups were determined as 5.7 and 2.3, respectively. Selective Hg2+-sensing properties have also been discussed, and log K-s values of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were found to show enhanced fluorescent intensity with a Hg2+ ion, demonstrating the potential application of RhOH for in vivo biological imaging.