Here, a novel rapid and ultrasensitive aptamer biosensor was designed for target-induced activation of AIE effect and followed by the activation of Crispr Cas12a (LbCpf1)-mediated cleavage to achieve dual-signal detection. The prepared DNA building blocks contain the target aptamer, ssDNA-Fc, and Activator1. In this system, the activation mode was divided into two steps. First, when the target interacts with the aptamers, the DNA building blocks would be disintegrated rapidly, releasing a mass of Ac1, generating ETTC-dsDNA aggregated to produce a fluorescence signal by the AIE effect. Second, with the release of Ac2, LbCpf1-crRNA was activated, which greatly improves the ssDNA-Fc cleavage efficiency to render signal amplification and ultrasensitive detection of the target. Satisfactorily, using this approach to detect gliotoxin, optimal conditions for detection was achieved for reducing the detection time to 55 min, achieving a low detection limit of 2.4 fM and a satisfactory linear in the range of 50 fM to 1 nM, which addressed the shortcoming of a weak electrochemical signal in previous sensors. The water-insoluble AIE material was coupled with DNA to obtain water-soluble ETTC-dsDNA and successfully introduced into the sensor system, with a low detection limit of 5.6 fM. Subsequently, the biosensor combined with handheld electrochemical workstation was successfully applied in the detection of gliotoxin in five actual samples, with a detection range of 32.0 to 2.09 × 108 pM. This strategy not only provides a novel and effective detection platform for mycotoxins in complex food matrices but also opens a promising avenue for various molecules detection in imaging and disease diagnosis.