This study was designed to determine oxolinic acid (OA) residues in the muscle of olive flounder (Paralichthysolivaceus) by a lateral flow immunoassay (LFIA) and ultra-performance liquid chromatography coupled with fluorescence detection (UPLC-FLD). After 25 cultured olive flounder were treated by dipping in water dosed with OA (50mg/L water for 1h), muscle tissues were sampled and prepared for the detection of OA residues. The visual detection limit of the test strip for OA determined by the C/T ratio of 1 was 28 mu g/kg of muscle. The detection limit of OA based on the standard curve was 4 and 1.6 mu g/kg of muscle using LFIA and UPLC-FLD, respectively, and recoveries of all spiked samples by LFIA and UPLC-FLD were >96%. LFIA and UPLC-FLD showed good correlation (R-2>.9997) for a series of samples collected over 5 days post treatment. The LFIA is applicable to the detection of OA in the muscle tissue of farmed fish, and is an effective and economical method that will enable high-throughput screening.