(Fe)-based single-atom catalysts (SACs), featuring atomically dispersed Fe atom active centers, display superior catalytic activity and selectivity. These excellent catalytic properties make Fe-SACs a promising candidate for construction of high-performance nanozymes, providing ample opportunities for developing biocatalytic medicine. It is of significant importance to systematically investigate the structure-controlled synthesis, property evaluation and mass-production of Fe-SACs with high loading. The emerging experimental and theoretical progress offers robust evidence to identify structure–activity relationship, thus shedding light on catalytic mechanisms toward specific catalytic reactions. In this review, we first summarize recent progress in synthesis methods and characterization of Fe-SACs, placing an emphasis on clarifying coordination environments of isolated Fe atoms anchored on supports. Then, we highlight the unique contribution of support materials on controlling active sites, optimizing catalytic activity, selectivity and chemical stability of Fe-SACs. To appreciate the latest innovation in nanotechnology, applications of Fe-SACs for biosensing and disease treatments are also discussed. Advancing the rapidly growing studies of SACs-related nanomedicine, we outline current challenges and perspective on future development of Fe-SACs. The intrinsic physiochemical and catalytic functions of Fe-SACs can assist us in designing more efficient catalysts at an atomic level in biological milieu.