In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg−1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.