Aflatoxin M₁ (AFM₁), one of the most toxic of the mycotoxins, is a global concern for feed and food contamination. A simple and fast aptasensor for the detection of AFM₁ was developed based on structure-switching signaling aptamer. The principle of the aptasensor is based on fluorescent signal change because of the formation of an AFM₁/aptamer complex. To construct the aptasensor, AFM₁ aptamers were modified with FAM and its complementary DNA (cDNA) was modified by TAMRA quenching group. Without adding AFM₁, AFM₁ aptamers hybridized with cDNA, resulting in quenching of the aptamer fluorescence due to the proximity of the fluorescent group of aptamer to the quenching group of cDNA. After adding AFM₁, the structure switch of AFM₁ aptamer was induced according to the formation of AFM₁/aptamer complex. The changes in the structure of the aptamer released the cDNA, resulting in fluorescence recovery of the aptamer, which enabled the quantitative detection of AFM₁ by monitoring the fluorescence enhancement. Under optimized conditions, this assay exhibited a linear response to AFM₁ in the range of 5–100 ng/mL with a detection limit down to 1.7 ng/mL. The assay was also applied to 2 brand infant formula rice flour samples spiked with a dilution series of AFM₁, obtaining satisfactory recoveries from 96.4 to 103.6% and 95–102.8%, respectively. The results demonstrated that this detection technique had a significant potential for high-throughput, and quantitative determination of mycotoxin levels in dairy products.