Single-atom nanozymes (SANs) are one of the newest generations of nanozymes, which have been greatly developed in the past few years and exploited widely for many applications, such as biosensing, disease diagnosis and therapy, bioimaging, and so on. SANs, possessing dispersed single-atom structures and a well-defined coordination environment, exhibit remarkable catalytic performance with both high activity and stability. In this paper, the most recent progress in SANs is reviewed in terms of their advanced synthesis, characterization, functional mechanisms, performance validation/optimization, and biomedical applications. Several technical challenges hindering practical applications of SANs are analyzed, and possible research directions are also proposed for overcoming the challenges.