The Edinburgh FLS920P steady-instantaneous fluorescence spectrometer was applied on the detection of the absorption and the emission spectra of ponceau 4R and amaranth, which are isomers to each other. After that, the spectral parameters of them were compared. Then, the density functional theory (DFT) and time-dependent density functional theory (TD-DFT) were used on the optimization of ponceau 4R and amaranth under the ground and excited state, respectively, in order to compare the differences in configurations of them under different states. On the base of the results above, the absorption and emission spectra of the two isomers were calculated with TD-DFT, and the polarized continuum model (PCM) was applied on the base of 6-311++G (d, p). The fluorescence mechanism, the relationships between the properties of fluorescence spectra and the molecular geometry were all analyzed. The results shows that, the structures of the two molecules are non-planar, these two naphthalene rings are not co-planar, respectively, and there’s hydrogen bond in amaranth. When the two isomers were on the ground state, the planarity of the naphthalene ring which exists the hydrogen bond mentioned above in amaranth is better than the corresponding part of ponceau 4R. The two isomers are nearly co-planar when they’re on the excited state. The molecular structures of ponceau 4R and amaranth optimized above are basically reasonable, for the quantum chemistry calculation spectral results are agree with the experiments. The planarity of the naphthalene rings on the right side in ponceau 4R is worse than that in amaranth, the ponceau 4R molecule experienced more vibration and rotation from the excited to the ground state, lost more energy, which lead to the reduction of energy for emitting fluorescent photons. So ponceau 4R has longer fluorescence emission wavelength than amaranth. In this paper, the molecular structure information of ponceau 4R and amaranth were obtained, and the differences of the spectral characteristics between them were found out. The results can provide references for the study of the relationship between the spectral properties and the configuration of isomers.