Objective Recently, single-atom nanozyme (SAN) has been widely studied due to its high atom utilization and various enzyme-mimicking activities. However, most of the SANs present low metal-atom densities, which restrict their further application and development. The aim of this study is to prepare SAN with high atomic loading and to systematically investigate its enzyme-like activities, in the hope of providing ideas for the preparation of highly loaded SANs and theoretical support for the application of SANs in a wider range of fields.Methods In this work, high-loading Cu SAN with metal contents up to 7.66% (w/w) was prepared using in-situ anchoring strategy. The metal salt precursors were confined into the carbon carriers consisting of aminated graphene quantum dots, and then urea was introduced to provide a rich N-coordination environment for Cu atoms. The chemical bond between Cu atoms and the carrier was further stabilized by pyrolysis under the protection of Ar gas. The low-loading Cu SAN used for comparison was prepared according to traditional metal organic framework (MOFs) anchoring method. Then, the peroxidase (POD)-like, oxidase (OXD)-like and superoxide dismutase (SOD)-like catalytic activities of high/low-loading Cu SAN were tested and compared with 3,3",5,5"- tetramethylbenzidine (TMB) and nitrotetrazolium blue chloride (NBT) as chromogenic substrates. The catalase (CAT)-like catalytic activities of high/low-loading Cu SAN were assessed by monitoring the decomposition rate of hydrogen peroxide (H₂O₂).Results The results suggested that the high-loading Cu SAN possesses the improved catalytic activity and selectivity towards POD-like and SOD-like activities, showing 3.4-fold and 8.88-fold higher than low-loading Cu SAN, respectively.Conclusion This work provides a strategy to synthesize SAN with high metal-atom density. And the results show that atomic regulation can be achieved by adjusting preparation methods which assist to obtain SAN with different catalytic selectivity, and lay foundation for the development and application of SAN in detection and sensing, diseases treatment and environmental protection.