A method for the characterization of the metabolite pentachlorophenol hydrogen sulfate (PCP-SO3H) of pentachlorophenol (PCP) in loaches was developed based on ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The loach samples exposed in low concentration of PCP solution were firstly crushed, then extracted by acetonitrile-water solution (70:30, v/v) containing 8% (v/v) triethylamine and purified by mixed anion exchange solid-phase extraction (SPE) cartridges. The chromatographic separation was carried out on a Waters ACQUITY BEH C18 column (100 mm×2.1 mm, 1.7 μm). The qualitative analysis of the metabolites of PCP was operated in a negative electrospray ion mode (ESI-) under full mass-data dependent MS2 (full mass-ddMS2) mode, and the data of quasi-molecular ion, isotope ions, and MS2 fragmentation ions of metabolites were obtained. The results revealed that the sulfonation was the main metabolic pathway for PCP in loaches, not the hydroxylation or glucuronate pathway. And the metabolite was found to be PCP-SO3H. Besides, with the increase of exposure time in PCP solution, the concentration of metabolite PCP-SO3H firstly increased; when the exposure time was up to 36 h, the concentration of metabolite in loach reached a maximum value; and then extending the exposure time, the concentration of PCP-SO3H gradually decreased. When the exposure time was up to 120 h, the concentration of PCP-SO3H in loaches reached a lowest value, and no significant change occurred for further time. The developed UPLC-HRMS method in this study could be used for the investigation of the metabolism of PCP in living beings.