Foodborne pathogenic bacteria are recognized as the main causes of microbial contamination in food safety. Early screening and ultrasensitive detection of foodborne pathogenic bacteria is critical procedure to guarantee food safety. Argonaute is emerging as a new tool for detection owing to the programmability and high specificity. We reported a Novel and One-step cleavage method based on Argonaute by integrating Tag-specific primer extension and Exonuclease I (Exo I) for the first time, termed as NOTE-Ago. In this method, the invA of Salmonella typhi and nuc gene of Staphylococcus aureus were amplified using Tag-specific primer and the remaining primers were digested by Exo I. Then amplicons were served as the guide DNA for PfAgo. Consequently, the fluorophore-quencher reporter could be cleaved via PfAgo, resulting in changes in fluorescent intensity. With this strategy, target nucleic acid could be dexterously converted into fluorescent signals. The NOTE-Ago assay could detect 1 CFU/mL with a dynamic range from 1 to 10⁸ CFU/mL. The satisfactory selectivity of NOTE-Ago assay further facilitated its application for detecting S. typhi- and S. aureus-contaminated food samples. This work enriches the toolbox of Argonaute-based detection and provides a one-step cleavage and rebuilding-free method for ultrasensitive detection of bacteria.