The rapid determination of oil concentration in water was investigated with three dimensional fluorescence spectra and parallel factor analysis (PARAFAC), and the influence of dissolved organic matter on the measurement of oil concentration was analyzed mainly. No. 0 diesel oil and humic acid was used for measuring sample in the experiment, the fast separation of No. 0 diesel oil fluorescence spectra from the overlapped spectrum of humic acid was studied through changing humic acid concentration, the contents of No. 0 diesel oil was obtained under different humic acid simulation conditions, the maximum average error was 1.67%, and the relative standard deviation was less than 1.29%. The results showed the possibility of oil concentration fast determination with a variation of dissolved organic matter in water based on three dimensional fluorescence spectra technique and PARAFAC.