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A rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for the detection of a widely used herbicide in rice cultivation, butachlor, in various polluted water samples. Three fluorescent tracers with different spacer arms and structures were synthesized to enhance the FPIA sensitivity. The effects of different fluorescent tracer structure and organic solvent concentration on the performance of the FPIA were investigated. The results showed that the fluorescent tracer 3-( 2) demonstrated the best FPIA sensitivity at pH 8.0, which remained at up to 20% methanol in a tolerance study. The IC 50 (50% inhibition concentration) of the FPIA was 207.0 ng mL À1 with the detection of butachlor ranging from 32.0 to 1218.0 ng mL À1 (at 20%-80% inhibition concentration), and the limit of detection (at 10% inhibition concentration) was 11.0 ng mL À1 . Recoveries, measured in spiked water samples, ranged from 75.1% to 134.5%. This newly developed FPIA was characterized by high specificity and reproducibility, allowing a rapid, low-cost, and sensitive determination of butachlor in polluted waters.