Nanozymes are a class of nanomaterials with enzyme-like catalytic properties, which have environmental tolerance and long-term stability. Improving catalytic activity and expanding the variety of nanozymes are the prerequisites to complement or even replace natural enzymes. The assembly of organic monolayer containing catalytic site on the surface of inorganic nanoparticles is a very effective strategy to improve catalytic activity and expand the variety of nanozymes. Here, we discuss how to construct organic monolayer on the surface of gold nanoparticles, classify the types of organic monolayer, and introduce their applications in nucleic acid hydrolysis and sensing. It is hoped to further promote the research progress of hydrolytic nanozymes.