Abstract: Mercury is one of the most threatening elements in the environment. The toxicity of different forms of mercury is different. Among them, methyl mercury is the most toxic one, which is harmful to human cardiovascular and cerebrovascular system, nervous system and so on. At the same time, methyl mercury will accumulate with the transmission of food chain. Fish is a common human food. Therefore, it is necessary to detect the mercury content of different forms in fish muscle simultaneously. It is of great research value to analyze the differences in different habitats and trophic levels. By changing the concentration of methanol in the mobile phase, negative high voltage of photomultiplier tube, current of hollow cathode lamp, reducing agent and carrier current, the separation degree and response value of different mercury speciation components were optimized. After optimized the experimental conditions, the fish muscle samples were treated by centrifugation, passing through the column, elution and other steps. Then the extract was added and centrifuged twice. After adjusting the pH, the extract was purified in a C18 column, and then eluted twice by the mobile phase. The fish samples were separated by liquid chromatography, and the three mercury forms in the extraction solution were detected by atomic fluorescence spectrometry. The results showed that the linear range of divalent mercury, methyl mercury and ethyl mercury was 0.2~10.0 μg/L respectively, the correlation coefficients were more than 0.999 9, the detection limit was 0.39~0.82 μg/kg, and the relative standard deviation (RSD) of parallel samples was less than 4.0%. The method is accurate, efficient, and is suitable for the speciation analysis of mercury in fish. If the method is applied to fish muscle samples from different habitats and different trophic levels, obvious differences of the results of different trophic levels and the correlation between the similar samples could be observed.