Chloramphenicol (CAP) is a banned substance in fish farming; yet, residues are sometimes detected in farmed fish products. Therefore, it is important that tissue distribution and elimination rates of CAP are measured to allow monitoring of fish farms. The tissue distribution and residue of CAP in carp (Cyprinus carpio L.) after muscle injection was investigated. Chloramphenicol concentrations were determined using high-performance liquid chromatography. Pharmacokinetics were used for the studies of CAP residue and the results showed that the CAP was easily assimilated in the liver, serum and gill, with the absorption rates of 6383, 3.02 and 1.39 h−1, respectively, following a single-dose injection of 80 mg kg bw−1. The elimination half-lives (t1/2) were 22.28, 15.47, 14.87, 9.28 and 5.32 h for the liver, serum, gill, muscle and kidney respectively. The t1/2 after 5 days of multi-doses of repeated CAP injection with 40 mg kg bw−1 was 1.09, 0.887, 0.872, 0.476 and 0.617 days for the five tissues respectively. The results validated that CAP in the liver, serum and gill could be markers for CAP residue analysis.