Abstract: With the continuous development of aquatic product production and processing, the safety risks posed by drug-resistant microorganisms and their genes have gradually attracted attention. Molecular sensors based on gene editing systems, especially Argonaute proteins that do not require protospacer adjacent motif (PAM) sites, have become an important direction in nucleic acid detection. To obtain the mesophilic Argonaute nuclease CbAgo (Clostridium butyricum Argonaute) and assess its feasibility in detecting human-derived drug-resistant opportunistic pathogens, the pET-28a/Rosetta(DE3) expression system was employed in this study. Soluble recombinant CbAgo protein was successfully expressed by induction with 0.1 mmol·L⁻¹ IPTG at 16 °C for 16 hours, followed by purification via nickel-affinity chromatography, yielding protein of approximately 87 kDa. The results showed that the CbAgo protein could specifically recognize and cleave single-stranded DNA under the guidance of guide DNA (gDNA). After further optimization of the reaction system, the cleavage and release of simulated single-stranded nucleic acid fragments of human-derived drug-resistant opportunistic pathogens were successfully achieved. Therefore, the CbAgo protein shows potential for application in subsequent detection of human-derived drug-resistant opportunistic pathogens in marine environments and gene editing research.