Aflatoxin B1 (AFB1) is a kind of potently carcinogenic fungal metabolite in food threatening human health, and it is crucial and challenging to develop advanced nonimmune approaches for AFB1 determination. Addressing this challenge, we successfully constructed a nanoassembly (PdE-PDI/SDS) by noncovalently coupling a cationic perylene diimide derivative (PdE-PDI) and sodium dodecyl sulfate (SDS), exhibiting high-density charges and a specific surface area for rapid sensing of AFB1. The large electronic conjugate structure and rigid plane of PdE-PDI enable it to form more stable σ-π, π-π coordination, and hydrogen bonds with AFB1. Additionally, the introduction of SDS significantly amplifies noncovalent interactions and enhances the quenching efficiency of PdE-PDI toward AFB1. The proposed PdE-PDI/SDS exhibited excellent specificity to AFB1 and showed dosage-sensitive detection with detection limit as low as 0.74 ng mL-1. Finally, the PdE-PDI/SDS was successfully applied in cereal samples with good recoveries from 94.61 to 109.92%. To our knowledge, this is the first time a fluorescent strategy from the point of self-assembly for AFB1 determination is reported, which holds great promise for wide applications of perylene diimide derivative in food safety.