The aim of this study was to evaluate the efficiency of fluorescence intensity measurement of PAH metabolites in fish bile (by Fixed Wavelength Fluorescence, FF) to study and assess PAHs contamination in aquatic organism. Nile tilapia (Oreochromis niloticus) was exposed to benzene, fluorene, anthracene, chrysene, and benzo[a]pyrene at non-lethal concentrations. The results showed that the fluorescence intensity and EROD activity were significantly increased with increase in exposure times and PAH concentrations. Moreover, the results of both methods had significantly positive relation. The maximum values were reached in 16 day after exposure. Thus, fluorescence intensity measurement by FF technique could be used as a potential tool for environmental study in the case of PAHs contamination in aquatic environment.