The food safety and medical health issues caused by pathogen are particularly prominent. The development of biosensing technologies is urgent to ensure pathogenic biosafety. Argonaute system, as a promising and cutting-edged next-generation nucleic acid test technology, has the potential to address the challenges faced by CRISPR/Cas system. In this review, we focused on the current state-of-art Argonaute-powered biosensing for pathogenic biosafety. First, we introduced current methods for nucleic acid testing and programmable nucleases, followed by the working principle of Argonaute system (PfAgo, TtAgo, CbAgo, etc). Then Argonaute-medicated nucleic acid biosensing was highlighted through amplification and amplification-free manners. In addition, we summarized the application of Argonaute tools in detecting bacteria, virus, mycoplasma, etc. Finally, we pointed out the challenges and perspectives. Current pathogen methods demonstrate low sensitivity and specificity, as well as lack capabilities for multiple and point-of-care testing. Recent studies have shown that Argonaute-powered biosensing is an innovative and rapidly growing technology that could significantly enhance detection capabilities for pathogen-related issues, addressing the limitations of current methods. The application of Argonaute-powered biosensing is both promising and desirable due to the potential to offer "customized" and streamlined detection in the field of pathogenic biosafety monitoring.