Nanozymes are nanomaterials with intrinsic natural enzyme-like catalytic properties. They have received extensive attention and have the potential to be an alternative to natural enzymes. Increasing the atom utilization rate of active centers in nanozymes has gradually become a concern of scientists. As the limit of designing nanozymes at the atomic level, single-atom nanozymes (SAzymes) have become the research frontier of the biomedical field recently because of their high atom utilization, well-defined active centers, and good natural enzyme mimicry. In this review, we first introduce the preparation of SAzymes through pyrolysis and defect engineering with regulated activity, then the characterization and surface modification methods of SAzymes are introduced. The possible influences of surface modification on the activity of SAzymes are discussed. Furthermore, we summarize the applications of SAzymes in the biomedical fields, especially in those of reactive oxygen species (ROS) scavenging and antibacterial. Finally, the challenges and opportunities of SAzymes are summarized and prospected.