Abstract: Objective: In this study, a colorimetric sensing system based on different aptamers and AuNPs was constructed for the rapid quantitative detection of aflatoxin M₁ (AFM₁) in milk. The detection performance of AFM₁ aptamers with 21 (A21) and 72 base (A72) lengths commonly used in literature reports was evaluated in actual samples. Methods: AuNPs solution was prepared by sodium citrate reduction. After adding AFM₁ aptamer and AFM₁ standard solution, the aptamer specifically combined with AFM₁ to form a special three-dimensional structure. With the addition of NaCl solution, the stability of AuNPs solution was disrupted and aggregation occurred, resulting in color change of the solution. AFM₁ was quantitatively detected by measuring the absorbance value and absorption spectrum of AuNPs solution. Results: Under the optimum condition, the limits of detection of AuNPs-aptasensor based on A21 and A72 aptamers were 25.26 μg/L and 5.77 μg/L, respectively, with the linear range of 10~800 μg/L. Moreover, the AuNPs-aptasensor had good selectivity and specificity. The recoveries of AuNPs-aptasensor based on A21 and A72 aptamers were 95.7%~103.6% and 94.3%~98.2%, respectively. Conclusion: The A72 performed better than A21 in detecting AFM₁ in milk, which would be related to the length, configuration and affinity of the aptamer. The colorimetric sensing method established based on aptamers A72 was simple, fast, sensitive, highly specific, and visualized, providing data reference for the application of AFM₁ aptamers in dairy products.