To explore the imaging effect of a melatonin-based molecular probe in glioblastoma. Methods: The probe Cy5-MT was prepared by coupling 5-methoxytryptamine with fluorescent dye Cy5. The in vitro MTT assay was used to evaluate the toxic effect of Cy5-MT on glioblastoma cell U87 and normal human glial cell HEB; Flow cytometry was used to evaluate the targeting ability of Cy5-MT to U87 and HEB; laser scanning confocal microscopy was used to evaluate the imaging effect of Cy5-MT on U87. Results: The structure of Cy5-MT was confirmed by mass spectrometry. In vitro cytotoxicity study showed that Cy5-MT had low toxicity to U87 and HEB, indicating high biosafety. Flow cytometry results showed that the percentage of cellular uptake and mean fluorescence intensity of the probe per U87 cells were significantly higher than the normal HEB cells. Imaging results showed that Cy5-MT can accurately image U87 tumor cells. Conclusion: The probe Cy5-MT can specifically target U87 glioblastoma cells to achieve accurate imaging of U87 and also demonstrates high biosafety.