According to the Lambert-Beer laws, the fluorescence spectra of oil in water were investigated by measuring excitation-emission matrixes with FS920 fluorescence spectrometer. On the basis of the three-dimensional fluorescence spectra of oil in water, the excitation wavelength of 290nm and emission wavelength of 324nm were chosen for the quantitative analysis of oil in water. The results show that the concentration of oil in water is linearly proportional to the fluorescence intensity in the range of 1ppm-100ppm, and the measurement model of concentration is obtained with the linear correlation coefficient of 0.9993. The artificial samples has also been tested and the results are satisfactory. The recovery is in the range of 92%-106%. The research provides experimental basis for the quantitative analysis of multi-component oil in water.