Single-atom nanozyme (SAzyme) is the hot topic of the current nanozyme research. Its intrinsic properties, such as high activity, stability, and low cost, present great substitutes to natural enzymes. Moreover, its fundamental characteristics, i.e., maximized atom utilizations and well-defined geometric and electronic structures, lead to higher catalytic activities and specificity than traditional nanozymes. SAzymes have been applied in many biomedical areas, such as anti-tumor therapy, biosensing, antibiosis, and anti-oxidation therapy. Here, we will discuss a series of representative examples of SAzymes categorized by their biomedical applications in this review. In the end, we will address the future opportunities and challenges SAzymes facing in their designs and applications.