Real-time monitoring the level of nitric oxide (NO) secreted by living cells is crucial for understanding the pathological and physiological processes in human body and related diseases. In this study, the COF-366-Fe/GA was prepared by in situ growth of COF-366-Fe on 3D graphene aerogel (GA), which integrated the highly electrocatalytic property generated by COF-366-Fe and the 3D porous structure possessed by GA to build a unique sensing component for ultrasensitive and real-time determination of NO. Benefiting from the remarkable synergy effect between COF-366-Fe and 3D GA, the prepared biosensor presents ultrasensitive response in the wide range of 0.18 to 400 μM with a lower detection limit (30 nM) and higher sensitivity (8.8 μA·μM^-1·cm^-2). Moreover, the molecular signals could be immediately captured by the fabricated device after release of the gas messenger NO secreted by human umbilical vein endothelial cells. These results imply that the proposed COF-366-Fe/GA with precisely controllable active sites and 3D porous structure solves randomly arrangement problem of active sites for the nitrogen-coordinated electrocatalyst, which would provide a promising powerful platform for real-time analysis of biomarkers in clinical diagnostics.