In recent years, three-dimensional fluorescence spectrometry has been widely used to study the transportation and transformation of the environment pollutants. But little understanding about the relationship between fluorescence characteristics and molecular structure restricts its application. In the present paper, the excitation-emission matrix (EEM) of the typical aromatic pollutants and isomers, phenanthrene and anthracene were studied. The result showed that there existed a peak locating at λex/λem=225/340 nm in the EEM of both phenanthrene and anthracene. Furthermore, the peaks at 275/360 nm of phenanthrene located quite close to the peak of anthracene at 285/360 nm. However, the difference between the EEM of phenanthrene and anthracene was significant. There existed the third fluorescence peak at 275/340 nm and the most intensive peak at 225/340 nm in the EEM of phenanthrene. The EEM of anthracene was more complicated. The most intensive peaks located at λex/λem=250/380, 250/400 and 250/425 nm respectiveoy. In addition, the fluorescence intensity of anthracene at 225/340 nm was about 1.63 times that of phenanthrene when their concentrations were about 0.058 1 mg·L-1. The orbital energy gap of the frontier molecules of phenanthrene and anthracene were 4.779 and 3.621 eV respectively according to the density functional theory. Owe to the smaller energy gap and better symmetry of electron cloud, anthracene was easier to be excited under the excitation of longer wavelength with higher fluorescence intensity. The density functional theory is a good tool to estimate the luminous capability of organic matters.