Here we demonstrate a noncompetitive idiometric nanobodies phage immunoassay for the determination of FB₁, utilizing two types of anti-idiotypic nanobody, that is specific for different epitopes within the hypervariable region of the primary mAb. Three alphatype anti-idiotypic nanobodies were obtained from a naive nanobodies phage-display library. A noncompetitive idiometric immunoassay was established with a combination of betatype anti-idiotypic nanobody and phage-displayed alphatype anti-idiotypic nanobody. The half-maximal saturation of the signal value (SC₅₀) was 0.68 ng/mL, with the limit of detection (LOD) was 0.19 ng/mL. When the noncompetitive assay was compared to the competitive ELISA (LOD = 3.41 ng/mL), an over 17-fold improvement in sensitivity was observed. A correlation (R²) was 0.988 between the data of the noncompetitive assay and LC–MS/MS for the determination of FB₁ in cereal samples. The noncompetitive idiometric immunoassay would have a broad utility for monitoring small molecules in food and environment. GRAPHICAL ABSTRACT