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Nitrogen-doped diamond-like carbon (DLC:N) films prepared by the filtered cathodic vacuum arc technology are functionalized with various chemical molecules including dopamine (DA), 3-Aminobenzeneboronic acid (APBA), and adenosine triphosphate (ATP), and the impacts of surface functionalities on the surface morphologies, compositions, microstructures, and cell compatibility of the DLC:N films are systematically investigated. We demonstrate that the surface groups of DLC:N have a significant effect on the surface and structural properties of the film. The activity of PC12 cells depends on the particular type of surface functional groups of DLC:N films regardless of surface roughness and wettability. Our research offers a novel way for designing functionalized carbon films as tailorable substrates for biosensors and biomedical engineering applications.