High performance liquid chromatography-tandem mass spectrometry of thiamphenicol in various tissues of Ictalurus punctatus was established in this study. This method has a linear correlation in the concentration range of 5-100 ng/mL, with a correlation coefficient of 0.998. The average recovery was 79.05%-95.58%, and the relative standard deviation was between 2.01% and 9.36%. The quantitative limits of thiamphenicol were 5.0 μg/L in plasma, and 5.0 μg /kg in muscle, skin, kidney, and liver. The pharmacokinetics of thiamphenicol in I. punctatus was investigated after oral administration with a single dose of 17.5 mg/kg body weight at the water temperature of 28 ℃. The concentration of thiamphenicol in each tissue was determined by high performance liquid chromatography-tandem mass spectrometry, and the data were processed by 3p97 pharmacokinetic software. The results showed that concentration-time data in plasma was described by a first-order absorption with two-compartment model. The T_peak was 8.00 h, the C_max was 933.75 μg/L, the AUC was 12.10 mg/L, the T_1/2β was 69.32 h and the T_1/2ka was 4.97 h. Pharmacokinetic results showed that thiamphenicol was distributed in a two-compartment model in the channel catfish, which was eliminated by first-order pharmacokinetics. The absorption, distribution and elimination of thiamphenicol in channel catfish were relatively fast. The study manifested that the mode of administration and precise administration dosing were also important in the control of channel catfish disease. The method is simple and reliable, and meets the requirements for the determination and pharmacokinetic study of thiamphenicol. The data are of great benefit for practical applications of thiamphenicol in channel catfish.