Accurate, specific, and cost-effective detection of toxic cyanogenic glycosides is crucial for ensuring biological health and food safety. In this study, a novel biosensor based on co-immobilized multi-enzyme system was constructed by artificial antibody-antigen-directed immobilization for the colorimetric detection of amygdalin through a cascade reaction catalyzed by β-glucosidase, glucose oxidase, and horseradish peroxidase. Artificial antibodies and antigens were prepared using catechol and 3,4-dihydroxybenzaldehyde, respectively, to generate mutual affinity recognition ability for enzyme immobilization. On this basis, the biosensing system showed a complete response to amygdalin within 4 min, with a linear range from 2 to 10 μM, a detection limit of 0.18 μM, and a quantification limit of 0.6 μM. In addition, this sensor had good precision, reproducibility, stability, and reusability. This study proposed a method for detecting cyanogenic glycosides, providing a successful case for the application of cascade biosensors in food safety detection.