To attain a circular carbon economy and resolve CO₂ electroreduction technology obstacles, single-atom catalysts (SACs) have emerged as a logical option for electrocatalysis because of their extraordinary catalytic activity. Among SACs, metal–organic frameworks (MOFs) have been recognized as promising support materials because of their exceptional ability to prevent metal aggregation. This study shows that atomically dispersed Ni single atoms on a precisely engineered MOF nanosheet display a high Faradaic efficiency of approximately 100% for CO formation in H-cell and three-compartment microfluidic flow-cell reactors and an excellent turnover frequency of 23,699 h⁻¹, validating their intrinsic catalytic potential. These results suggest that crystallographic variations affect the abundant vacancy sites on the MOF nanosheets, which are linked to the evaporation of Zn-containing organic linkers during pyrolysis. Furthermore, using X-ray absorption spectroscopy and density functional theory calculations, a comprehensive investigation of the unsaturated atomic coordination environments and the underlying mechanism involving CO* preadsorbed sites as initial states was possible and provided valuable insights.