Bilirubin plays a significant role in human health management, particularly in the case of jaundice. Because of the need for the monitoring of bilirubin levels in jaundice patients, the development of a robust sensitive method becomes essential. Here, we describe the development of a highly sensitive and selective turn-off fluorometric detection method for bilirubin in blood serum samples using nitrogen-doped carbon dots (N-CDs). N-CDs was synthesized by the pyrolysis process, using citric acid and L-asparagine as the carbon and nitrogen sources, respectively. The prepared N-CDs solution showed highly intense blue emission with good stability. The HR-TEM image of N-CDs revealed spherical dot-like structures with an average size calculated to be 7.16 nm. Further, the surface functional groups of N-CDs were analyzed by FT-IR, Raman, XRD, and XPS techniques. Fluorescence spectra showed the maximum emission intensity at 443 nm (λ