Influenza A virus RNA promoter region has recently attracted much attention as RNA targets for the development of novel anti-influenza drugs. However, there are very limited reports on small RNA-binding ligands targeting this region. In this work, we report that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, exhibits the strong and selective binding to the internal loop structure of the influenza A virus RNA promoter. This binding accompanies the remarkable light-up response of TO-PRO-3 in the deep-red spectral region. By virtue of these binding and fluorescence signaling functions, TO-PRO-3 can work as a useful indicator for the assessment of the binding capabilities of various test compounds for this RNA region with a view toward the development of anti-influenza drug candidates.