The catalysis of chemical transformation is usually thought to be mediated by small organic molecules, transition metal complexes, or protein enzymes. However, the pioneering discovery of self-splicing intron-splicing RNA ribozymes demonstrated that nucleic acids can also promote catalysis, with efficiency comparable to that of proteases. In 1994, the discovery of deoxyribonase further broadened the understanding of the catalytic function of nucleic acids. In addition, some nucleic acid-mediated nanomaterials also have the function of mimicking natural enzyme activity. The discovery of the catalytic function of nucleic acid not only breaks the traditional cognition that nucleic acid is only genetic material, but also extends the function of nucleic acid to the field of artificial enzymes. In this chapter, the above nucleic acids with catalytic functions are defined as functional nucleases. Functional nucleases refer to nucleic acids or nucleic acid complexes that have special structure/morphology and participate in the catalytic functions of cutting, linking, linking, color rendering, and luminescence of nucleic and nonnucleic substrates. Then, eight types of functional nucleases are introduced: ribozyme, DNA-cleaving enzyme, DNA mismatch linkase, aptamer cleaving enzyme, G-quadruplex peroxidase, functional nucleic acid nanozyme, DNA metallization nanozyme, nucleic acid coordination nanozyme. In addition, the applications of these enzymes were summarized, and the future development trend and application prospect of functional nucleases were also prospected.