Enrofloxacin is one of the normally used flouroquinolones in mammalian and fish but its withdrawal time and studies were remain obscure. The residual contents of enrofloxacin in fish muscle were analyzed by using HPLC-FLD. More than 0.1 mg/kg of ENR was detected in muscle tissues and the residues were found over 1 year after treatment. The concentration of ENR in Paralichthys olivaceus was not affected by water temperature and lasted for an extended amount of time. The spike recoveries of ENR in the muscle tissue ranged from 78% to 85%. From this results, we need the prescription by veterinarian or aquatic organism disease inspector in ENR usage to assure safety of fish. Future research is required to determine the recommendation dose of ENR for side effects and safety.