The present paper investigates the influence of solvent purity on the test of three-dimensional fluorescence spectra, determines the process of its pre-purification, and studies the influence of the concentrations on the three-dimensional fluorescence characteristic indexes such as the characteristic peaks position, fluorescence intensity(F), and ratio (R) of fluorescence intensity when the phenomena of fluorescence quenching won’t appear. The result shows that the concentration affects the value F and value R largely but its effect on the relative peak intensity (Rs) is relatively small. Different crude oils has different value Rs. According to those characteristics, this paper presents that the value Rs can be used as a new characteristic index for identification of crude oil. This conclusion was confirmed by testing many crude oils stably and reliably from different areas with their concentrations at 5 mg·L_-1. This method can be applied to identify most crude oils preliminary and further accurate analysis can be carried out together with other index.