<p>Consumption of edible insects is common in non-Western countries of Africa, Asia, Oceania, and Latin America. However, their consumption has significantly increased in Europe in recent years thanks to their remarkable nutritional properties. Edible insects provide a valuable source of high-quality proteins, fats, minerals, and vitamins. Nevertheless, the absence of global regulatory guidelines poses a risk associated with their consumption due to the potential presence of pathogens and contaminants, such as mycotoxins, which are toxic compounds produced by fungi and represent a major threat to food and feed safety. Our approach integrates advanced nanobiotechnology to develop fluorescent antibodies by conjugating a recombinant superfolder green fluorescent protein (sfGFP) with a single-chain antibody (scFv). This fusion allows for precise detection of the immune complex formed between the HT-2 toxin and a biotinylated anti-HT-2 antibody. Additionally, we employed advanced computational tools, including AlphaFold and MOE, to deepen our understanding of the binding interactions present in the immune complex, confirming the strong interaction between the Fab/HT-2 toxin immunocomplex and the scFv antibody fragment, in contrast to the weaker binding observed with the Fab/T-2 toxin and the scFv. The method demonstrates high sensitivity, with an EC₅₀ of 10.3 ± 0.6 ng mL⁻¹, a dynamic range of 3.4 ± 0.1 to 31 ± 3 ng mL⁻¹, a limit of detection of 0.43 ng mL⁻¹, and a limit of quantification of 1.2 ng mL⁻¹ in buffer&#xa0;solution. The assay exhibited excellent precision, with a reproducibility of 4% and no cross-reactivity with other mycotoxins. Application to contaminated cricket flour yielded recoveries between 91 and 133%, with coefficients of variation from 6 to 13%. These results indicate that the developed immunoassay is highly sensitive, selective, and reliable for detecting HT-2 toxin in food matrices, providing a promising tool for mycotoxin screening in food safety.</p> Graphical Abstract <p></p>