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Amoxicillin (AMX) is widely used for the treatment of gastrointestinal and systemic infections. AMX residues above certain levels may pose a threat to human health. Therefore, this study aimed to understand the pharmacokinetics of AMX in Japanese eel, Anguilla japonica, using high-performance liquid chromatography (HPLC) with fluorescence detection. Japanese eels were treated with AMX at concentrations of 40 or 80 mg kg−1 body weight (BW) daily via oral administration. The results revealed that the recoveries of AMX from the muscle, liver, and sera of eels were 84.7–105.1%, 81.8–104.9%, and 88.6–106.3%, respectively. The observed peak retention time was approximately 4.2 min. After administration of 40 mg kg−1 BW per day of AMX, the pharmacokinetic parameters were detected, respectively. The average drug residue concentration in muscles was 180 and 260 ng mL−1 on day 1 and day 2, respectively, and in the liver, it was 70 and 110 ng mL−1 on day 1 and day 2, respectively, after the 40 mg kg−1 BW daily AMX administration. Residue concentrations in the muscle and liver on day 3 after 40 mg kg−1 BW administrations were below the limit of detection (