In this article we have investigated intermolecular excited-state proton transfer (ESPT) of fireflys chromophore D-luciferin in DMSOwater binary mixtures using steady-state and time-resolved fluorescence spectroscopy. The unusual behavior of DMSOwater binary mixture as reported by Bagchi et al. (J. Phys. Chem. B 2010, 114, 1287512882) was also found using D-luciferin as intermolecular ESPT probe. The binary mixture has given evidence of its anomalous nature at low mole fractions of DMSO (below X-D = 0.4) in our systematic investigation. Upon excitation of neutral D-luciferin molecule, dual fluorescence emissions (protonated and deprotonated form) are observed in DMSOwater binary mixture. A clear isoemissive point in the time-resolved area normalized emission spectra further indicates two emissive species in the excited state of D-luciferin in DMSOwater binary mixture. DMSOwater binary mixtures of different compositions are fascinating hydrogen bonding systems. Therefore, we have observed unusual changes in the fluorescence emission intensity, fluorescence quantum yield, and fluorescence lifetime of more hydrogen bonding sensitive anionic form of D-luciferin in low DMSO content of DMSOwater binary mixture.