The steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra of riboflavin in water, DMSO and chloroform were measured by British Edinburgh FLS920P spectrometer. It was found that the absorption peaks hardly change in different solvents, and the fluorescence peaks show a significant red shift with the increase of the polarity of solvents, because the charge distribution of the excited state leads to the difference between the absorption and fluorescence spectra. Besides, the fluorescence lifetime of riboflavin in water is also longer than that in the other two solvents, which can be attributed to the hydrogen bond interaction between riboflavin and water. Furthermore, the ground and excited states of riboflavin molecules in different solvents were calculated by using Gaussian 09 software. Through frontier molecular orbital analysis, the stimulated transition of riboflavin is the transition of π electrons on benzene ring and nitrogen heterocycle ring to the antibonding orbital π* of benzene ring and CN, CO conjugated double bond. The results show that the charge distribution of riboflavin increases the dipole moment and the solute-solvent intermolecular interaction, which leads to spectral variation and a red shift in fluorescence spectra. Finally, the effect of H-bond on the spectra of riboflavin was considered by the molecular electrostatic potential and noncovalent interaction analysis. The theoretical absorption and emission peaks of the polymer structure are closer to the experimental results, which shows that the results of the polymer are reasonable. The ring structure formed by water dimer and riboflavin can increase the rigidity of riboflavin molecule, which is beneficial to the fluorescence emission, thus decreasing the probability of non-radiative transition and prolonging the fluorescence lifetime.