In this work, we present an advanced fluorescence assay for the detection of traces of ocratoxin A and neomycin in food. The described assay is based on measurement of the fluctuations of the fluorescein-labeled analytes by a focused laser beam in the absence and in the presence of the specific antibodies anti-analytes. A competitive assay based on the utilization of unlabeled analytes was developed. The obtained results indicated that the combination of high-avidity IgG antibodies together with an innovative fluorescence immunoassay strategy resulted in the detection limit of 0.0078 ng and 0.0156 ng for ochratoxin A and neomycin, respectively.