Three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy was applied to characterize the mineral oil in ethanol. In the paper, a simple way of treating the first-order Rayleigh, Raman and second-order Rayleigh scatter is used involving subtracting an EEM of a solvent blank, and removing and replacing the values with zeros. The corrected results showed that the characteristic three-dimensional fluorescence spectra of mineral oil were as follows: there was one mainly peak in the fluorescence spectrum of the kerosene, and the peak was identified at excitation/emission wavelengths (Ex/Em) of 270/290 nm; there were two intense fluorescence peaks in the fluorescence spectrum of 0# diesel, located at (Ex/Em) of 240/344 nm and 270/362 nm respectively; there were several fluorescence peaks in the fluorescence spectrum of lubricating oil, with the two intense peaks located at (Ex/Em) of 240/348 nm and 258/358 nm respectively. Furthermore, the relation between the relative fluorescence intensity and the concentration of mineral oil was studied, and the sensitivity and the limitation of detection of the method were also analyzed. The results of this study show that the method of three-dimensional fluorescence can be used to detect the low concentration of oil.