Recently, single-atom nanozymes have made significant progress in the fields of sterilization and treatment, but their catalytic performance as substitutes for natural enzymes and drugs is far from satisfactory. Here, a method is reported to improve enzyme activity by adjusting the spatial position of a single-atom site on the nanoplatforms. Two types of Cu single-atom site nanozymes are synthesized in the interlayer (Cu /PHI) and in-plane (Cu /PHI) of poly (heptazine imide) (PHI) through different synthesis pathways. Experimental and theoretical analysis indicates that the interlayer position of PHI can effectively adjust the coordination number, coordination bond length, and electronic structure of Cu single atoms compared to the in-plane position, thereby promoting photoinduced electron migration and O activation, enabling effective generate reactive oxygen species (ROS). Under visible light irradiation, the photocatalytic bactericidal activity of Cu /PHI against aureus is ≈100%, achieving the same antibacterial effect as antibiotics, after 10 min of low-dose light exposure and 2 h of incubation.