The Supercritical Fluid Extraction (SFE) technique was used to fractionate Suwannee River reference fulvic acid (FA). The fractions were characterized by gas chromatography (GC) and reversed-phase high performance liquid chromatography (RP-HPLC). A SFE fractionation method was developed using stepwise gradient of supercritical CO₂ and methanol. Three FA fractions were separated. The average mass recovery was 102% with the coefficient of variation of 2.8%. The fractionation dynamics and the difference in the ratios of UV absorption to fluorescence emission indicate the real fractionation of the FA. The HPLC chromatographic peak patterns and the spectra of the corresponding peaks were almost indistinguishable. The overall results of this research support the argument that FA exhibits polymer-like molecular structure.