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Herein, we developed a multi-analyte fluorescence immunoassay for detection of three organophosphate pesticides (triazophos, parathion, and chlorpyrifos) in various agro-products (rice, wheat, cucumber, cabbage, and apple) using fluorescently labeled oligonucleotide and gold nanoparticle (AuNP) signal amplification technology. The AuNP probes for the three analytes were constructed by simultaneously modifying the corresponding antibodies and fluorescently labeled oligonucleotides on the probe surface. Three fluorophores (6-FAM, Cy3, and Texas red) with high fluorescence intensity and little overlap of excitation/emission wavelengths were selected. The method showed satisfactory linearity for triazophos, parathion, and chlorpyrifos in the ranges of 0.01–20, 0.05–50, and 0.5–1000 μg/L, respectively. For the 3 analytes, the limits of detection (LODs) were 0.007, 0.009, and 0.087 μg/L, respectively. The …