An efficient method based on high-performance liquid chromatography coupled with atomic fluorescence spectrometry (HPLC-AFS) was successfully developed for the simultaneous determination of four mercury species including Hg2+, methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) in water. Samples were enriched and cleaned up with a solid-phase extraction (SPE) pretreatment using a thiol cartridge. Some key parameters including the selection of a SPE cartridge, eluent type, eluent volume, and interference factors were systematically investigated. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of methanol and 60 mmol L-1 ammonium acetate with 10 mmol L-1 L-cysteine by gradient elution. Under the optimized conditions, good linearity (r ≥ 0.9991) was observed between 0.20 to 10.0 μg L-1. The limits of detection were in the range of 0.001 - 0.002 μg L-1. High recoveries (87.2 to 111%) and good reproducibility (1.1 - 6.5%) were obtained. Such a method is sensitive, selective and accurate, which can be applied to the quantification of mercury species in water samples.