Single‐atom nanozymes (SAzymes) showcase not only uniformly dispersed active sites but also meticulously engineered coordination structures. These intricate architectures bestow upon them an exceptional catalytic prowess, thereby captivating numerous minds and heralding a new era of possibilities in the biomedical landscape. Tuning the microstructure of SAzymes on the atomic scale is a key factor in designing targeted SAzymes with desirable functions. In this review, we firstly discuss and summarize three strategies for designing SAzymes and their impact on reactivity in biocatalysis. The effects of choices of carrier, different synthesis method, coordination modulation of first/second shell, and the type and number of metal active centers on the enzyme‐like catalytic activity are unraveled. Next, we make a first attempt to summarize the biological applications of SAzymes in tumor therapy, biosensing, antimicrobial, anti‐inflammatory and other biological applications from different mechanisms. Finally, how SAzymes are designed and regulated for further realization of diverse biological applications is reviewed and prospected. We envisage that the comprehensive review presented within this exegesis will furnish novel perspectives and profound revelations regarding the biomedical applications of SAzymes.This article is protected by copyright. All rights reserved