Nowadays, single-atom nanozymes (SAzymes) and single-atom catalysts (SACs) have flourished in the field of catalysis owing to their high catalytic performance and exceptional atom utilization efficiency, thereby enhancing biosensing capabilities. In comparison to natural enzymes, SAzymes offer several advantages, including cost-effectiveness, ease of production, and robust catalytic activity, making them highly promising for biosensing applications. Notably, SAzymes demonstrate superior catalytic efficiency and selectivity compared with traditional nanozymes. In this context, this review delineates the enzyme-like characteristics of SAzymes aimed at enhancing food safety, with a focus on the primary factors that influence their catalytic efficacy. The discussion has been expanded to include the use of SAzymes for screening various pesticide residues, particularly organophosphate pesticides (OPPs), carbamates, acetamiprid, pyrethroids, and other pesticide types, which are present in agricultural food products. These applications are realized because of the exceptional properties of single-atom structures, including enhanced reaction kinetics, high active site density, and tunable electronic properties. The integration of SAzymes into sensing platforms holds great potential for the development of cost-effective, sensitive, and reliable tools for the real-time monitoring of pesticide residues. Finally, this paper highlights the current challenges and outlines potential opportunities for the advancement of SAzyme-based biosensing technologies.