Pharmacokinetics of florfenicol were studied in tissues from Pelodiscus sinensis Japanese strain using high performance liquid chromatography-tandem mass spectrometry. One hundred fifty healthy soft-shell turtles with the weight of 250±50 g were adnubustered florfenicol at a dosage of 30 mg/kg for 7 consecutive days under the temperature of 25.0±2.0℃. After the last feeding, tissues including blood, muscle, liver and kidney were collected at 1 h, 2 h, 4 h, 8 h, 16 h,24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 240 h and 360 h, respectively. Florfenicols in different tissues were extracted with ethyl acetate, cleaned by hexane, and detected by HPLC-MS/MS with negative electrospray ionization under multiple reaction monitoring. Quantitative analysis was used by internal standard method. Results showed that: The peak times of concentrations of florfenicol in plasma, muscle, liver and kidney were 4 h, 8 h, 8 h and 8 h, with the maximum content of 167 μg/kg, 188 μg/kg, 67.15 μg/kg and 85.71 μg/kg, respectively. The elimination half life was14.9 h, 9.4 h, 29.5 h and 13.0 h. The concentration-time curves of florfenicol in blood, liver, muscle and kidney were: C_blood=210.332 e^{-0.070 6 t} +938.161 e^{-0.046 5 t}, C_muscle=5 642.635 e^{-0.077 1 t}+5 765.891 e^{-0.074 4 t}, C_liver=111.596 e^{-0.036 0 t}+4.339 e^{-0.023 5 t} and C_kidney=176.509 e^{-0.0654 t}+615.697 e^{-0.053 6 t}, respectively. Florfenicol was absorbed and eliminated fast in P.sinensis Japanese strain. The withdraw period should not be less than 15 d under this experiment condition.