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Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B 1 (FB 1 ). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB 1 detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB 1 detection, with a dynamic range from 7.3 to 22.6 ng mL -1 , a detection limit of 1.1 ng mL -1 , and IC 50 value of 12.9 ng mL -1 , which was significantly lower than the IC 50 value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B 1 and B 2 , as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB 1 2000 μg kg -1 and 103% for FB 1 4000 μg kg -1 ), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.