Assignment of the fluorescence spectrum of firefly luciferin in aqueous solutions was achieved by utilizing not only emission energies but also theoretical absorption spectra and relative concentrations as estimated by pKa values. Calculated Gibbs free energies were utilized to estimate pKa values. These pKa values were then corrected by employing the experimental results. It was previously thought that the main peak near 550 nm observed in the experimental fluorescence spectra at all pH values corresponds to emission from the first excited state of the luciferin dianion [Ando et al. (2010) Jpn. J. Appl. Phys. 49, 117002-117008]. However, we found that the peak near 550 nm at low pH corresponds to emission from the first excited state of the phenolate monoanion of luciferin. Furthermore, we found that the causes of the red fluorescence at pH 1-2 are not only the emission from phenol monoanion but also the emission from the protonated species at nitrogen atom in the thiazoline ring of dianion.