Suwannee River natural organic matter (SRNOM) was treated by 7 M urea and then purified by dialysis on 10 kDa membrane. The untreated SRNOM and treated (USRNOM) samples were examined using UV–visible and fluorescence spectroscopies and reversed-phase high-performance liquid chromatography (RP-HPLC) with online absorbance and fluorescence detection. USRNOM was 1.5-fold more absorbing at 280 nm than SRNOM and four fold less fluorescent than SRNOM upon excitation at 270 nm. RP-HPLC analyses of the two samples revealed that USRNOM was somewhat more hydrophobic than SRNOM and both samples contained at least two groups of HS-like fluorophores with different hydrophobicity and protein-like fluorophore(s). Data indicate that protein-like fluorophores were not lost during dialysis. They showed hydrophobic properties and seemed highly fluorescent. HS-like and protein-like fluorophores from water NOM could be successfully separated by RP-HPLC. This raises the prospect of their further research and identification and could be significant for future NOM chemical structure characterization.