The residue levels of ampicillin (AM) in cultured olive flounder, Paralichthys olivaceus (average 300g) at 20±1.0℃were studied by oral, intramuscular and dipping administration (routes). The concentrations of AM in the plasma were determined by HPLC-UV detector. The average recoveries of AM in spiked samples between 0.01∼10 ppm were ranging from 84.45% to 91.26% for plasma. The limit of detection for AM was 0.05 ppm by using this method.