Single-atom catalysts (SACs) are considered one of the promising strategies to achieve efficient energy conversion, due to their advantage of both maximum atomic utilization and minimum catalyst cost. However, finding a balance between increasing the atom loading and preventing the agglomeration of metal single atoms is a current research hotspot. Herein, we have developed single-atom cobalt embedded in N-doped carbon nanoboxes as high-efficiency bifunctional electrocatalysts for overall water splitting in alkaline/acidic electrolytes. Due to the reliable metal-nonmetal bonds between the Co single atom and the substrate carbon, where the uniformly dispersed Co atoms could be effectively and stably riveted on the carbon nanoboxes, and the as-prepared electrocatalyst possesses high mass loading of single Co atoms (∼10.2 wt%). In addition, the systematic X-ray absorption fine structure (XAFS) and density functional theory (DFT) calculations were performed to further investigate the relationship between coordination number, configuration and electrocatalytic properties of Co single atoms and N atoms. Based on the above results, the presence of Co atoms induces the formation of pyrr-N and possesses Co@CNB-N4 configuration with HER overpotential of 45 mV, comparable to Pt/C (20 wt%). As for OER, Co@CNB-N4 still has satisfactory catalytic performance, superior to the benchmark catalyst RuO2. Thus, this work builds a bridge to understand the impact of metal single atoms and substrate configuration on catalytic performance and opens a door to the successful synthesis of SACs with high loading of non-precious metal atoms, high atomic utilization and electrocatalytic activity.