A rapid detection method for the pesticide pentachlorophenol (PCP) — polarization fluoroimmunoassay (PFIA) — in the dynamic range of 10–9,000 ppb was developed. PCP may form polychlorinated dibenzo-p-dioxins, making environmental monitoring of this compound an issue of great importance. In order to optimize the PFIA procedure, a number of fluorescein-labeled PCP derivatives and similar compounds (tracers) were synthesized, and the influence of their structure on PFIA characteristics was studied. Also, two antisera were tested in developing PFIA for PCP. The developed method is highly specific for PCP and can be used for its determination in water samples at a level down to 10 ppb. Total time of the assay for 10 samples is about 7 min. The assay provides a useful and a highly practical screening tool for the processing of large numbers of samples and for the preliminary estimation of potential dioxins contamination in water resources.