The residue levels of ampicillin (AM) in cultured olive flounder, Paralichthys olivaceus (average 300g) at $20{\pm}1.0^{\circ}C$ were studied by oral, intramuscular and dipping administration (routes). The concentrations of AM in the plasma were determined by HPLC-UV detector. The average recoveries of AM in spiked samples between 0.01~10 ppm were ranging from 84.45% to 91.26% for plasma. The limit of detection for AM was 0.05 ppm by using this method. Plasma concentrations of AM were determined after oral dosage (10, 20 and 40 mg/kg body weight), intramuscular injection (5, 10 and 20 mg/kg body weight) and dipping (10, 20 and 40 ppm; 1 h). Samples were taken at 1, 3, 5, 10, 15, 24, 30, 48, 96, 144, 216, 264 and 360 h post-administration. In oral dosage of 10, 20 and 40 mg/kg, it's peak concentrations were $3.62{\pm}0.97$, $5.20{\pm}0.70$ and $11.18{\pm}0.87{\mu}g/ml$, respectively at 10 h post-administration, but AM was not measurable at 144, 360 and 360 h post-administration, respectively. In intramuscular injection of 5, 10 and 20 mg/kg, it's peak concentrations were $6.92{\pm}1.29{\mu}g/m1$, $9.89{\pm}2.22{\mu}g/ml$ and $19.85{\pm}2.97{\mu}g/ml$, respectively at 5 h post-administration, but AM was not measurable at 216, 264 and 264 h post-administration, respectively. In dipping of 10, 20 and 40 ppm, it's peak concentrations were $4.39{\pm}1.10$, $9.57{\pm}1.51$ and $11.61{\pm}1.92{\mu}g/ml$, respectively at 3 h post-administration, but AM was not measurable at 264, 264 and 360 h post-administration, respectively. Therefore, the plasma distribution and elimination levels of AM in olive flounder were dosage-dependant manner in all administration routes.