Single-atom catalysts (SACs), in which the metal active sites are isolated on the support and stabilized by coordinated atoms such as oxygen, nitrogen, sulfur, etc., represent the maximum usage efficiency of the metal atoms. Benefiting from the recent progress in synthetic strategies, characterization methods, and computational models, many SACs that deliver an impressive catalytic performance for a variety of reactions have been developed. The catalytic selectivity and activity are critical issues that need to be optimized and augmented in the areas of nanotechnology and biomedicine. This review summarizes some recent experimental and theoretical progress aimed at clarifying the structure of SACs and how they influence the catalytic performance. The examples described here elaborate on the utility of SACs and highlight the strengths of these catalysts in the applications of biomedicine, environmental protection, and energy conversion. Finally, some current challenges and future perspectives for SACs are also discussed.