Abstract: The pharmacokinetics of drug in animal was influenced by different environmental factors. In order to explore the effect of temperature and water velocity on pharmacokinetics of drug in rainbow trout, three temperature (5 °C, 10 °C, 15 °C) and three water velocity (8 cm/s, 16 cm/s 24 cm/s) were studied. The temperature and water velocity were controlled by the control system, samples were determined by high-performance liquid chromatography (HPLC). Results showed that at temperatures of 5 °C, 10 °C and 15 °C, T_max was 8.67 h, 4.78 h and 2.39 h; T_1/2α was 0.86 h, 0.80 h and 0.77 h; T_1/2β was 49.18 h, 45.81 h and 38.35 h; AUC was 140.49 μg/(L·h), 130.4 μg/(L·h) and 112.78 μg/(L·h), respectively. Metabolism of enrofloxacin in rainbow trout showed: in a certain temperature range, the rate of drug absorption and distribution at higher temperature is greater than that at lower temperature; the time to peak was shorter at higher temperature. At water velocity of 8 cm/s, 16 cm/s and 24 cm/s, T_max was 8.57 h, 6.03 h and 4.04 h; T_1/2α was 5.47 h, 2.16 h and 0.27 h; T_1/2β was 26.54 h, 6.93 h and 2.13 h; T_1/2ka was 7.68 h, 5.00 h and 2.01 h. The rate of drug absorption and distribution at higher water velocity is greater than that at lower water velocity; the time to peak was shorter at higher water velocity. In conclusion, the results from the present study indicated that in a certain range, the increase of temperature and water velocity improved the rate of drug absorption and distribution of enrofloxacin in rainbow trout.