A method for rapid determination of pentachlorophenol in fishery water was established by surface-enhanced Raman spectroscopy (SERS). The concentrated gold colloid was prepared to use as Raman substrate according to the classic method, and the optimal SERS enhancement effects were obtained by adjusting the pH of the system, optimizing the coagulants, nanoparticles volume and adsorption time. The method had a good linearity between the peak intensity and the pentachlorophenol concentration in the range of 0.2～4.0 μg/mL, and the detection limit was 0.5 μg/mL. The recovery ranged from 78.74% to 112.30%, and the relative standard deviation was between 6.1%～11.3%. The sensitivity and specificity of the SERS method were over 95%, and the rates of false positive and false negative results were less than 5%, which were all conform to the evaluation specification of food rapid analysis method. The method is fast, accurate and easy to operate, and it provides a good foundation for the rapid detection of pentachlorophenol in fishery water.