Developing convenient and sensitive detection methods for antibiotic residues in food is beneficial for ensuring food quality and human health. The tough challenges that limit the development of sensitive, quantitative, portable, on-the-spot antibiotic detectors are the lack of simple and effective target recognition and signal amplification strategies, and direct digital quantification. Herein, we developed a visual digital quantitative aptasensor, based on a binding-induced 3-D DNA nanomachine signal probe, for the simple and sensitive, on-the-spot detection of antibiotics.