Nanozymes, a class of nanomaterials mimicking the function of enzymes, have aroused much attention as the candidate in diverse fields with the arbitrarily tunable features owing to the diversity of crystalline nanostructures, composition, and surface configurations. However, the uncertainty of their active sites and the lower intrinsic deficiencies of nanomaterial-initiated catalysis compared with the natural enzymes promote the pursuing of alternatives by imitating the biological active centers. Single-atom nanozymes (SAzymes) maximize the atom utilization with the well-defined structure, providing an important bridge to investigate mechanism and the relationship between structure and catalytic activity. They have risen as the new burgeoning alternative to the natural enzyme from in vitro bioanalytical tool to in vivo therapy owing to the flexible atomic engineering structure. Here, focus is mainly on the three parts. First, a detailed overview of single-atom catalyst synthesis strategies including bottom-up and top-down approaches is given. Then, according to the structural feature of single-atom nanocatalysts, the influence factors such as central metal atom, coordination number, heteroatom doping, and the metal-support interaction are discussed and the representative biological applications (including antibacterial/antiviral performance, cancer therapy, and biosensing) are highlighted. In the end, the future perspective and challenge facing are demonstrated.