A highly sensitive protocol for signaling norepinephrine (NEP) in human fluids and neuronal cell line models should be established for clinical investigation of some neuronal diseases. A metal-free electrode catalyst was designed based on a sulfur-doped carbon spheroidal surface (S-CSN) and employed as a transducing element for selective signaling of NEP in biological samples. The designed electrode of S-CSN features a spherical construct and curvature surface to form a spheroidal nanolayer with an average layer size of <2 nm. S-CSN shows surface topography of a circular surface curvature with a rugged surface texture, ridge ends, and free open spaces between interlayers. The rich-space diversity surfaces offer highly active surface with facile molecular/electron diffusion, multi-diffusive centers, and high target loading along with in-/out-of-plane circular spheres of the S-CSN surface. The active doping of S atoms onto the carbon-based electrode creates an active transducing element with many active sites, strong binding to targeted molecules, facile diffusion of charges/molecules, long-term durability, and dense reactive exposure sites for signaling NEP at ultratrace levels. S-CSN could be a sensitive and selective nanosensor for signaling NEP and establishing a sensing protocol with high stability and reproducibility. The sensory protocol based on S-CSN exhibits high sensitivity and selectivity with a low detection limit of 0.001 μM and a wide linear range of 0.01-0.8 μM. The in vitro sensory protocol for NEP secreted from living cells (neuronal cell line model) under stimulated agents possesses high sensitivity, low cytotoxicity, and high biocompatibility. These results confirm the successful establishment of NEP sensor in human blood samples and neuronal cells for clinical investigation.