Abstract: Aflatoxin B₁ (AFB₁) has carcinogenic, teratogenic and mutagenic effects. It has been categorized as a group I carcinogen by the international agency for research on cancer (IARC). Thus, AFB₁ poses a great threat to consumers' health. When Tb³⁺ was alone in solution, its characteristic fluorescence could not be detected. Nevertheless, the single-stranded oligonucleotides can greatly enhance the emission of Tb³⁺ in solution. The target AFB₁ can bind to aptamer, thus causing conformational change of aptamer to form a double chain. The duplexes can significantly inhibit the emission of Tb³⁺. Based on above principles, this study constructed a fluorescence characteristic of rare earth terbium ion (Tb³⁺) unlabeled nucleic acid aptamer sensor can realize homogeneous and rapid detection of AFB₁. According to this principle, this method achieves quantitative analysis of AFB₁ via the change of fluorescence signal. The LOD was 1.84 ng/mL. Due to the specificity of nucleic acid aptamer in target molecule recognition, AFB1 and other mycotoxins can be accurately identified and detected in complex matrix. Moreover, the recovery rate obtained by nucleic acid aptamer sensor in the experiment was 95.94%~107.12%, which verified the effectiveness of this method. This method does not need label aptamers so as to save cost. The whole process of detection is easy to operate without separation procedures. The detection can be completed in a clean centrifuge tube, which can realize homogeneous analysis of AFB₁.which can realize homogeneous analysis of AFB₁.