This work introduces an extremely easy method for preparing luminescent carbon dots (CDs) at ambient temperature using 1,2-naphthoquinone sulphonate and ethylenediamine as precursors via self-exothermic reaction without energy input. The as-obtained CDs have a high quantum yield (34.1 %), a production yield of 21.2 %, and a small size diameter (3.44 nm). Various techniques (NMR, TEM, EDX-mapping, XPS, XRD, FT-IR, fluorescence, and UV–visible spectroscopy) were used to characterize the prepared CDs. The CDs exhibited an excitation-independent emission with λex of 275 nm, demonstrating their homogeneity and high purity. The anticancer drug vincristine (VCR) quantitively quenched the fluorescent signal of the synthesized CDs, allowing their application as the first fluorescent nano-sensor to determine VCR. The quenching effect was linear within the range of 0.2–5.0 μg mL−1, enabling the determination of VCR in vials, plasma, and for content uniformity testing with a detection limit of 0.06 μg mL−1. Moreover, the synthesized CDs were employed as a bio-sensing platform to detect VCR in cancer cells owing to their good selectivity, excellent biocompatibility, minimal cytotoxicity, and high stability. The fabrication of CDs with excellent properties at room temperature under mild conditions paves the way for new advancements in the room temperature synthesis of CDs and offers a highly efficient alternative to traditional synthesis approaches.