Perylene diimide derivatives were used as probes to investigate the effect of the molecular structures on the fluorescence quenching mechanism in a perylene diimide/graphene oxide system. The electrons transferred from the excited state of dyes to the conductive band of graphene oxide with different concentrations were determined by fluorescence spectra. The results indicated that the quenching efficiency of perylene diimides by graphene oxide was not only dependent on the difference between the lowest unoccupied molecular orbital level of dyes and the conduction band of the graphene oxide, but also mainly on the difference in the molecular structures.