In　situ　measurement　of　nitric　oxide　(NO)　in　living　tissue　and　single　cells　is　highly　important　for　achieving　a　profound　comprehension　of　cellular　functionalities　and　facilitating　the　precise　diagnosis　of　critical　diseases;　however,　the　progress　is　greatly　hindered　by　the　weak　affinity　of　ultratrace　concentration　NO　in　cellular　environment　toward　electrocatalysts.　Herein,　a　new　strategy　is　reported　for　precisely　constructing　orbital　coupled　dual-atomic　sites　to　enhance　the　affinity　between　the　metal　atomic　sites　and　NO　on　a　class　of　N-doped　hollow　carbon　matrix　dual-atomic　sites　Co─Ni　(Co1Ni1-NC)　for　greatly　boosting　electrocatalytic　NO　performance.　The　as-synthesized　Co1Ni1-NC　demonstrates　a　substantially　higher　current　density　than　Ni1-NC　and　Co1-NC,　coupled　with　exceptional　stability　with　a　negligible　degradation　rate　of　0.6　µA·cm−2·h−1,　which　is　the　best　among　the　state-of-the-art　electrocatalysts　for　NO　oxidation.　Experimental　and　theoretical　investigations　collectively　reveal　that　the　pivotal　role　of　d-d　orbit　coupling　between　Co　and　Ni　sites　enables　Ni　to　acquire　additional　electrons,　leading　to　the　occupation　of　Ni＇s　3dxy/yz　within　the　2π　orbitals　of　NO,　thus　weakening　the　N≡O　triple　bond　and　concurrently　accelerating　NO　adsorption　kinetics.　It　is　demonstrated　that　Co1Ni1-NC-coated　nanoelectrode　can　achieve　the　in　situ　sensing　of　NO　in　living　organs　and　single　cells.　©　2024　Wiley-VCH　GmbH.