In this paper, Fe3O4@SiO2@γ-mercaptopropyltrimethoxysilane (γ-MPTS) magnetic nanoparticles was prepared and a new method of magnetic solid phase extraction (MSPE)-high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICP-MS) was developed for the speciation of mercury including inorganic mercury (Hg2+), methylmercury (MeHg+) and phenylmercury (PhHg+) in environmental water, wastewater, tap water and fish samples. A rapid separation of three target mercury species was achieved in 8min by employing relatively high ratio of methanol in HPLC mobile phase. Various parameters affecting Fe3O4@SiO2@γ-MPTS-based MSPE of target mercury species have been investigated. Under the optimized conditions, the limits of detection for Hg2+, MeHg+ and PhHg+ were in the range of 0.49–0.74ngL−1. The intra- and inter-day relative standard deviations (n=5) were less than 9.0% and 12%, respectively. The developed MSPE-HPLC-ICP-MS method was validated by the speciation of mercury in the Certified Reference Material of DORM-2 dogfish as well as real-world samples including environmental water, wastewater, tap water and fish samples, and it has the advantages of simple operation, rapid separation, high sensitivity, high enrichment factor and is suitable for the analysis of mercury species in samples with complex matrix. Display omitted •A method of MSPE-HPLC-ICP-MS was proposed for the speciation of Hg2+, MeHg+ and PhHg+.•HPLC separation of Hg2+, MeHg+ and PhHg+ was completed within 8min.•The developed method provided high EFs, short analysis time, low LODs and wide linear range.•It is suitable for speciation of trace/ultra-trace mercury in real samples with complex matrix.