The effect of a water-miscible room-temperature ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF4]), on the behavior of a common and popular probe fluorescein is assessed. Depending on solubilizing milieu and conditions, fluorescein may exist in one or more of its prototropic forms (cationic, neutral (zwitterionic, quinoid, and lactone), monoanionic (phenolate and carboxylate), and dianionic) in the solution. Information regarding the various prototropic forms of fluorescein in buffer-rich aqueous IL mixtures ([bmim][BF4] <= 50 wt %, mole fraction <0.1) at different pH is obtained via UV-vis absorbance and fluorescence. The addition of [bmim][BF4] is found to cause lactonization of fluorescein in the solution. Hydrolytic instability of IL [bmim][BF4] is observed to be much lower than that of NaBF4. Absorbance and fluorescence from fluorescein are observed to be insignificant when dissolved in neat [bmim][BF4], suggesting the presence of predominantly the lactone form. The IL [bmim][BF4] behaves similar to apolar media in this context. It is demonstrated that various prototropic forms of fluorescein may be generated within [bmim][BF4] by the addition of buffer of appropriate pH. Significant bathochromic shift in absorbance and fluorescence band maxima of dianionic fluorescein as concentration of [bmim][BF4] is increased correlates well with the decrease in the hydrogen-bond-donating acidity of the medium. The lack of good linear correlation between the Stokes shift and the orientational polarizability suggests the possible presence of specific Coulombic interaction between the IL cation and the fluorescein dianion within the mixture. It is established that IL [bmim][BF4] has an interesting and unique effect on the prototropic behavior of fluorescein.