Fluoroquinolones are the most common group of antibacterial agents currently used in the Korean aquaculture industry, and use of these agents has been increasing steadily. High performance liquid chromatography (HPLC) with fluorescence detection was used for the simultaneous determination of five fluoroquinolones in fish and shellfish: ofloxacin (OFL), pefloxacin (PEF), norfloxacin (NOR), ciprofloxacin (CIP), and enrofloxacin (ENRO). Fish and shellfish muscle was homogenized, and protein, lipid, and low molecular weight pigments were then excluded from the homogenate. The final eluates were analyzed by HPLC equipped with a Shiseido UG-120 type C18 reverse-phase column ($4.6{ imes}250 mm$, $5{mu}m$) and a fluorescence detector (excitation at 280 nm, emission at 450 nm). The mobile phase was 0.1 M phosphoric acid and acetonitrile solution (91:9, v/v) and tetrahydrofuran (THF) was added to it at a rate of 5 mL per a liter of the mobile phase. Adequate chromatography separation was obtained using the above method. Average recoveries of fortified samples at levels from 0.05 to 0.5 mg/kg were $72.3{pm}2.5-84.5{pm}1.2%$ for OFL, $82.7{pm}3.3- 109.3{pm}7.5%$ for NOR, $85.3{pm}6.6-116.0{pm}7.9%$ for PEF, $76.0{pm}4.3-109.3{pm}12.4%$ for CIP, and $78.7{pm}5.9-100.0{pm}9.8%$ for ENRO. The limit of detection of OFL was $5{mu}g/L$, the others were $1{mu}g/L$. We concluded that the new analytical method was suitable for the determination of fluoroquinolones in fish and shellfish.