Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.