A simple and highly sensitive immunoassay based on a competitive binding and bio-barcode amplification was designed for detection of small molecules, triazophos. The gold nanoparticles (AuNPs) were modified with monoclonal antibodies and 6-carboxyfluorescein labeled single-stranded thiol-oligonucleotides (6-FAM-SH-ssDNAs); the fluorescence of 6-FAM was quenched by AuNPs. Ovalbumin-linked haptens were coated on the bottom of microplate to compete with the triazophos in the sample for binding to the antibodies on the AuNP probes. The fluorescence intensity was inversely proportional to analyte concentration. Parameters of AuNP probes preparation and immune reaction were optimized. At the optimal conditions, the salting process was shortened to 1 h and 166 ± 9 ssDNAs were loaded onto a single AuNP. The competitive fluorescence bio-barcode immunoassay was performed on water, rice, cucumber, cabbage and apple samples. The linear range of the method was 0.01-20 μg Land the limit of detection (LOD) was 6 ng L. The recovery and relative standard deviations (RSDs) ranged from 85.0 to 110.3% and 9.4-17.4%, respectively. Good correlations were obtained between the results of the developed method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, it is suggested that the competitive fluorescent bio-barcode immunoassay had the potential to be used as a sensitive method for detection of a variety of small molecules in various samples.