The study was aimed at investigating the pharmacokinetics of amoxicillin trihydrate (AMOX) in olive flounder (Paralichthys olivaceus) following oral, intramuscular, and intravenous administration, using high-performance liquid chromatography following. The maximum plasma concentration (Cmax ), following oral administration of 40 and 80 mg/kg body weight (b.w.), AMOX was 1.14 (Tmax , 1.7 h) and 0.76 μg/mL (Tmax , 1.6 h), respectively. Intramuscular administration of 30 and 60 mg/kg of AMOX resulted in Cmax values of 4 and 4.3 μg/mL, respectively, with the corresponding Tmax values of 29 and 38 h. Intravenous administration of 6 mg/kg AMOX resulted in a Cmax of 9 μg/mL 2 h after administration. Following oral administration of 40 and 80 mg/kg AMOX, area under the curve (AUC) values were 52.257 and 41.219 μg/mL·h, respectively. Intramuscular 30 and 60 mg/kg doses resulted in AUC values of 370.274 and 453.655 μg/mL·h, respectively, while the AUC following intravenous administration was 86.274 μg/mL·h. AMOX bioavailability was calculated to be 9% and 3.6% following oral administration of 40 and 80 mg/kg, respectively, and the corresponding values following intramuscular administration were 86% and 53%. In conclusion, this study demonstrated high bioavailability of AMOX following oral administration in olive flounder.