Prokaryotic Argonautes （pAgos） are programmable nucleases involved in cellular defense against foreign DNA invasion. In vitro， pAgos can bind to small single stranded guide nucleic acid （ssDNA/ssRNA） to recognize and cleave complementary DNA/RNA. In vivo， pAgos preferentially target multiple copies of genetic elements， bacteriophages， and plasmids， thereby inhibiting the propagation of invading nucleic acids and bacteriophage infections. Prokaryotic Argonautes （pAgos）， as an emerging class of programmable nucleases， are more flexible than the most widely used CRISPR Cas system and has shown great potential in biotechnology. Previous studies were primarily focused on the thermophilic pAgos. Nowadays， the main applications based on thermophilic pAgos include molecular diagnosis and DNA assembly in vitro. Researchers have gradually shifted their focus to pAgos from mesophilic biological sources to promote the application of Ago-based biotechnology in vivo， such as gene editing. Although genome editing has not yet been achieved by pAgos， it’s possible to develop the next generation techniques for genome-editing based on pAgos with more and more pAgos being discovered and researchers studying the catalytic mechanism of pAgos in depth. This article summarized the known representative pAgos and biotechnologies based on the development of pAgos. The challenges and potential strategies faced by the application of pAgos in prokaryotic and eukaryotic organisms were briefly analyzed.