The objective of this study was to investigate the early biological response in the olive flounder exposed to sub-lethal concentrations of waterborne phenanthrene (0.5, 1 or 2 μM). The fish were exposed for 4 weeks and we analyzed their enzymatic defense system, antioxidant and phase II enzyme activities, to evaluate the chronic exposure toxicity of phenanthrene. Waterborne phenanthrene affected antioxidant enzymes and glutathione-mediated detoxification as an enzyme defense system. Hepatic, gill and kidney glutathione reductase as well as glutathione S-transferase, and catalase activities were markedly elevated after two or four weeks of exposure. These enzyme activities of the olive flounder, Paralichthys olivaceus, seem to be a convenient approach for monitoring pollution in coastal areas against polycyclic aromatic hydrocarbon pollution including phenanthrene.