Sudan dye is generally used as a coloring agent in textile and cosmetic industries. However, Sudan dye exhibits potentials to cause cancer, toxicity, and allergy because of its metabolites including amines and naphthol related substances. Despite banning Sudan dye, the Sudan dye is used by many unscrupulous food manufacturers for enhancing food appearance. This paper introduces a new aptamer based on Sudan III detection methods. An in vitro selection and amplification method named SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is employed to screen out Sudan III specific aptamer. The desired aptamer is obtained after 12 rounds of screening and cloning. To establish aptamer affinity and specificity for Sudan III, ELONA (enzyme‐linked oligonucleotide assay) and dot blot analyses are performed. ELONA assay indicates that 100 nM is the optimum concentration of the aptamer for Sudan III detection, while the minimum detection limit of Sudan III is 4 ng/well. The aptamer exhibits stable secondary structure with the stem‐loop and G‐quadruplex in the main structure. The selected single ssDNA aptamer C07 demonstrates binding affinity and the highest specificity to Sudan III (C07: KD = 22.37 ± 3.943 nmol L−1). Based on the results, a good correlation is observed between aptamer C07 and Sudan III and it is concluded that the new Sudan III specific aptamer has potential to be applied for rapid and accurate detection of the Sudan III in food products. Practical Applications: Examination of the chili sauce sample in a simulation experiment indicates that the aptamer C07 can be applied to rapid detection in foods. The nucleic acid aptamers‐based food detection method has the advantages of high sensitivity, specificity, easy operation, short detection time, and low cost. It can realize the rapid detection of illegal additives in foods, which has a great significance in the field of food safety research. The SELEX method is used to select a new Sudan III specific aptamer. ELONA and Dot blot assays further suggest that aptamer C07 can be used for selective detection of Sudan III. Therefore, the detection in chili sauce samples offers feasibility for more complex samples.

α-amanitin is a polypeptide isolated from the fruiting body of Amanita exitialis. It is the main toxin in wild mushrooms and toxic, often lethal, in animal and humans. In this study, the artificial nucleic acid aptamers targeting α-amanitin were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in vitro, in order to develop an analytical tool for α-amanitin detection. The specificity of aptamer H06 with α-amanitin was confirmed using Enzyme-Linked OligoNucleotide Assay (ELONA) and Dot blot, and no non-specific was observed. Based on the ELONA platform, the minimum detectable concentration of aptamer H06 for α-amanitin was 8 ng/mL. The circular dichroism (CD) spectroscopy experiment indicated aptamer H06 forms a stem-loop and intramolecular G-quadruplex and it can stable exist in binding buffer and PBS buffer. Moreover , the affinity test showed a strong binding force between α-amanitin and the aptamer H06, with the dissociation constant (KD) of 37.5±5.135 nM. And the accurary of the ELONA assay based on aptame H06 was demonstrated in real mushroom samples. In summary, our data could demonstrate a possibility of the development of apta-based diagnostic platform and detection method for α-amanitin.