Carbon-based single-atom catalysts (SACs) with atomic sizes of active sites have become the promising candidates for a variety of catalytic systems because of their high atom utilization, and unique electronic structures. Different types of single-atom sites can be fabricated via multiple preparation strategies, which would demonstrate distinct different coordination configurations and electronic features, and ultimately affected the structure-catalysis relationship of SACs in targeted reactions. As a result, it is necessary to identify the active sites of SACs and understand the structure-catalysis relationship of SACs at the atomic scale. In this review, a variety of preparation strategies of carbon-based SACs were documented. Then, the recent development on versatile characterization techniques and computational achievements were summarized regarding in understanding the electronic and geometric characteristics of carbon-based SACs. Finally, major challenges and development directions concerning single-atom sites identification and advanced tools development are discussed to shed light on future research of carbon-based SACs. Display omitted Understanding the coordination configurations and electronic features of the SACs help to understand their structure-catalysis relationship in targeted reactions at the atomic scale