Three-dimensional fluorescence spectroscopy has become a hot topic in the identification of oil spills by many researchers because of its high sensitivity, good selectivity, simple operation and analysis for multi-component mixtures. However, the Rayleigh scattering in the three-dimensional fluorescence spectrum will have a great influence on the accurate detection of the spectrum, so it is of great significance to eliminate the influence of the Rayleigh scattering effectively for the qualitative identification and quantitative analysis of the spectrum. In this paper, the instrument calibration method, background subtraction method, Delaunay triangle interpolation method and Missing Data Recovery (MDR) method were used to correct the Rayleigh scattering in the three-dimensional fluorescence spectrum of the oil spill. Firstly, the seawater SDS micelle solution was used as a solvent, the jet fuel and the lube were mixed according to different relative volume fraction ratios to prepare eight calibration samples and three test samples. Then, the three-dimensional fluorescence spectra of 11 samples were determined by FS920 steady-state fluorescence spectrometer. Moreover, the interference of Rayleigh scattering was eliminated by instrument calibration method, background subtraction method, the Delaunay triangle interpolation method and missing data recovery (MDR) method respectively. Then the kernel consensus diagnosis method was used to estimate the optimal number of components. Finally, the PARAFAC was used to qualitatively identify and quantify the three-dimensional fluorescence spectrum data of the mixed oil samples. The results show that the instrument calibration method using the emission wavelength lag excitation wavelength to eliminate Rayleigh scattering will lose part of the effective spectral information. The background subtraction method cannot completely eliminate the Rayleigh scattering, and there is still scattering interference in the spectrum. The excitation and emission spectra obtained by PARAFAC will be distorted, and the predicted concentration value deviation is large. After the Rayleigh scattering is eliminated by Delaunay triangle interpolation method, the excitation and emission spectra obtained by PARAFAC have a higher agreement with the real spectrum, and the predicted concentration value deviation is small. However,after the Rayleigh scattering is eliminated by MDR, the excitation and emission spectra obtained by PARAFAC analysis have the highest agreement with the real spectrum, and the predicted concentration value deviation is the smallest of these methods, and the sample recovery rate is 98.9% and 100% respectively, the RMSEP is limited to less than 0.130. According to the results of qualitative identification and quantitative analysis, MDR can effectively eliminate the influence of Rayleigh scattering on the basis of ensuring that the original characteristic spectrum is not distorted. It is an ideal method to eliminate Rayleigh scattering in the three-dimensional fluorescence spectrum.