Since the discovery of the RNA functions modulating gene expression, functional RNA has become an attractive drug target. Ligand-RNA interactions have been studied by covalent labeling of either the ligand or RNA with fluorescent dyes or immobilization on the sensing devices. However, it is desirable to investigate the interactions without any chemical modifications in order to keep the native secondary and tertiary structures. Displacement assays satisfy this criterion because neither covalent labeling nor immobilization is necessary for the assay. We here report a fluorescent indicator displacement assay for the investigation ora ligand-RNA interaction that exploits a novel fluorescent indicator, a 2,7-distributed 9H-xanthen-9-one derivative. The assay was found to be applicable for screening new ligands binding to RRE RNA from chemical library.