<i>Pf</i> Ago‐Enabled Fluorescence Biosensing: Recent Advances and Future Perspectives

Accurate, rapid, and cost‐effective nucleic acid detection is critical for disease diagnosis, food safety, and environmental monitoring. In recent years, prokaryotic Argonaute proteins (pAgos) have emerged as powerful tools for nucleic acid analysis due to their programmable endonuclease activity. Among them, Pf Ago, derived from the hyper‐thermophile Pyrococcus furiosus , has garnered significant attention for its ability to utilize small DNA guides (gDNAs) to cleave DNA targets at high temperatures with high specificity, independent of Protospacer Adjacent Motif (PAM) sequences. This review systematically summarizes the discovery history, structural domains, and catalytic mechanism of Pf Ago as a DNA‐guided DNA endonuclease. Furthermore, recent applications in pathogen screening, single‐nucleotide variant (SNV) genotyping, antibiotic resistance analysis, and non‐nucleic acid target detection are reviewed. Finally, current limitations, such as high‐temperature requirements and multiplexing challenges, are discussed alongside future perspectives, aiming to provide insights for the development of next‐generation point‐of‐care testing (POCT) platforms.