Three-dimensional (3D) excitation-emission matrix (EEM) fluorescence spectroscopy is applied to characterize the coal oil. The results show that the 3D fluorescence spectra of coal oil in aqueous solution mainly have one broad peak. This peak is identified at the excitation/emission wavelengths of 270/290 nm. The relation between the fluorescence intensity and the concentration of coal oil is also studied. When the concentration lies between 2-2000 ppm, the relation between the fluorescence intensity and the concentration of coal oil is well linear. The nature of solvents significantly affects the EEM fluorescence of coal oil.