To find out the impact of different saturation filling in the treatment process of bioretention systems, composition and dynamics of dissolved organic matter in runoff were analyzed using three-dimensional excitation emission matrix fluorescence spectroscopy (EEM). The results indicated that the major organic compounds of interstitial water were microbial metabolites from sponge iron, fulvic-like substances from brick bat and volcanic, and aromatic protein-like substances, fulvic-like substances and biometabolism from chippings. After 48 h treatment, fluorescence peakof interstitial water associated with fulvic-like and brick bat were weakened, but volcanic and chippings were strengthened. The best purification of organic substance was obtained by sponge iron at 84.52%, followed by volcanic and brick bat at 77.25% and 77.90% respectively, and chippings at 29.20%. Thevolume fluorescence integral of fulvic-like substances in effluent of bioretention decreased to 58.04% on average, but aromatic protein-like substances increased on average to 65.36%. HIX of all samples were smaller than 4, which means the organic compounds mainly come from microorganism supersession activity. Aromatic protein-like substances degraded by microbes easily, which could promote microorganism activity and nitrogen removal by denitrification. The design suggests that sponge iron and brick bat would be better as the saturation filling. External carbon source would be able to choose organic compounds which are easily utilized by microbe.