Simulated weathering experiments on crude oil were carried out to investigate the changes in the fluorescence fingerprint characteristics of crude oil caused by weathering processes. The excitation-emission matrix fluorescence spectra of crude oil were analyzed in the peak-ratio method and principal component analysis. The research results showed that weathering had no effect on the fluorescence spectra fingerprinting characters, but had effect on the relative intensity of peaks, whose changing direction was determined by peak location. Changes of the peak intensity ratios and the score of Principle Component 1(PC1) were separated in two stages (on the water surface or in the water), which might be brought about by different weathering mechanisms. Peak intensity ratios and the score of PC1 could be simultaneously used to determine the extent of weathering in oil fingerprint identification.