Objective To establish a method for the determination of three mercury speciation in fish samples by liquid chromatogram-inductively coupled plasma mass spectrometry (LC-ICP-MS) with ultrasound-assisted acid extraction procedure. Methods The sample was extracted with acid under ultrasonic condition. The extraction efficiency of different hydrochloric acid concentration was tested and compared. The chromatographic separation conditions and the operation parameters of ICP-MS were optimized. Results The content of three species in fish samples was determined. Relative standard deviations were less than 5%. The recoveries were in the range of 82%-99%. The detection limit was 6.2 μg/kg. Conclusion The result indicated that the method was accurate, sensitive, simple, stable and was suitable for the determination of trace mercury speciation in fish.