The development of natural molecule-derived carbon nano dots (CNDs) marks a significant advancement in biocompatible and sustainable nanomaterials. Tryptophan, capable of crossing the blood-brain barrier (BBB), serves as a precursor to numerous pharmacologically active compounds, while isatin and its derivatives have demonstrated anti-tumor effects, including against brain cancers. This study aimed to synthesize fluorescent CNDs from tryptophan-isatin hybrid precursor and explore their applications in glioblastoma treatment. These CNDs were characterized using techniques such as TEM, SEM-EDS, FTIR, XPS, Raman spectroscopy and UV-Vis spectrophotometry. In vitro tests using the U-87 glioblastoma cell line evaluated cell viability, affinity, and BBB permeability. The CNDs, between 4 and 7 nm in size, exhibited blue and green fluorescence, with no cytotoxic effects observed at concentrations up to 25 µg/mL. The highest BBB permeability rate was determined as 4.3 × 10⁻⁵ cm/s. Additionally, the CNDs demonstrated radiotherapeutic properties, leading to a 51 % reduction in cell viability. This research contributes to nanomedicine by introducing a novel biocompatible material with potential for targeted brain cancer imaging and therapy, while also suggesting broader applications beyond glioblastoma.