In the present study three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy was applied to characterize the dissolved organic matter (DOM) in the overlying water under aerobic and anaerobic condition. The effects of redox condition were significant on 3DEEM fluorescence spectra of DOM, and in the aerobic condition, the peak intensities of protein-like fluorophores were both higher than those of the humic-like fluorophores, however, the phenomenon of the oxidative degradation of humic-original DOM could be seen. While in anaerobic condition, the peak intensities of the humic-like fluorescence were increased with increasing the incubation time. After the 21 day anaerobic incubation, the peak intensities of the humic-like fluorescence can be as 3.51 and 3.78 times higher than those of protein-like fluorescence. The differences in the DOM fluorescence parameters, e.g., peak intensities, locations and fluorescence index, indicate the difference in the chemical structures and various origins of the DOM in the overlying water between sediment-water interfaces.