Using high performance liquid chromatography with tandem mass spectrometric (HPLC-MS/MS), we established a new method for detecting florfenicol and florfenicol amine which enabled us to study florfenicol, related metabolites (florfenicol amine) pharmacokinetics and tissue distribution of Acipenser baeri after oral administration of florvenicol in vivo. In this study, oral administration of florfenicol was performed on the Acipenser baeri at a dosage of 15 mg/kg body weight at 22 ℃, and the blood plasma, liver, kidney and muscle were then sampled to measure the concentration of florfenicol and its metabolite (florfenicol amine) using HPLC MS/MS method. The results indicated that the concentration time course of florfenicol and florfenicol amine can be described by a two compartment open model with first order absorption in vivo. The maximal concentration（C_max）of florfenicol in blood plasma was 3.4 μg/mL. Time point of maximal concentration（T_peak）was 2.943 h. Volume of distribution（V/F）was 3.267 L/kg. Half life of elimination（t_1/2β）was 31.21 h. Area under the time concentration curve(AUC) was 76.51 μg·h/mL, C_max（FFA）/ C_max（FF）and AUC_FFA/AUC_FF were 5.44%, 20.73%;Florfenicol and florfenicol amine were found to be extensively distributed and had a similar regularity in several tissues, with a higher concentration in liver and kidney. The results indicate that florfenicol have following features in Acipenser baeri: rapidly absorbed, high peak concentration, slow elimination and wide range of tissue distribution, and mainly metabolized or eliminated in the form of initial drugs.