Enrofloxacin (ENRO) is one of the most commonly used fluoroquinolones for treating bacterial disease in olive flounder (Paralichthys olivaceus) farming, but its withdrawal time for industrial-scale farming has not been well established. Withdrawal times of ENRO following oral administration were evaluated in olive flounder under field conditions. Fish were held in an inland fish tank and fed a commercial mediated diet containing 5 mg/kg of ENRO for 9 days. Seven fish per sampling point were examined during and after treatment. ENRO and its major metabolite, ciprofloxacin (CIP), were analyzed using high-performance liquid chromatography with a fluorescence detector. The concentration of ENRO and CIP in muscle increased during the medication period, and then decreased rapidly The sum of ENRO and CIP concentration in olive flounder peaked on day 6, with a maximal concentration in muscle of 4.30 mg/kg. ENRO residues were eliminated rapidly; at 10 days post treatment, the level in muscle was 0.10 mg/kg, but it took about 50 days to be reduced to below 0.1 mg/kg. After 60 days, the residual concentration was below 0.1 mg/kg in all samples. The level of ENRO accumulation at the beginning of oral administration was variable, according to the farming conditions, but the overall exhaustion time was almost the same. We concluded that an adequate withdrawal period of enrofloxacin is 60 days in the case of oral administration.