Abstract: Melon is one of the most typical vegetables and cash crops to fully meet the ever-increasing demand for fruit products in recent years. Among them, grafting treatment can be used to improve the quality and yield of watermelon. Grafting healing has been one of the key steps to the survival and quality of watermelon seedlings. The graft success can be represented by the reconnection of the vascular bundle between the scion and rootstock. In this study, the fluorescence tracer materials were prepared with nitrogen and sulfur-doped carbon dots (N, S-CDs) with stable fluorescence characteristics and non-toxic performance. Hyperspectral fluorescence imaging was then used to monitor the healing process of watermelon grafted seedlings. Firstly, the N, S-CDs were prepared at 330-630 nm excitation for the outstanding fluorescence emission, as well as the red and blue double emission characteristics. Secondly, the watermelon seedlings 1-8 days after grafting were labeled with the carbon dot staining, and then placed in a dark room, where the labeled images were collected with a high spectrometer. Finally, the hyperspectral map was processed to extract the data. The principal component map was also analyzed for the spectral data. The results showed that there was no connection between scion and rootstock when the N, S-CDs were used for fluorescence tracing. The carbon point substances failed to transfer into the scion through the xylem, leading to no observation at the scion site. By contrast, the scion was connected to the rootstock after the healing of grafted seedlings. The carbon points were then transferred to the scion through the xylem, where the fluorescence was observed at the scion site. The hyperspectral imaging technique can be expected to quickly, automatically and non-destructively identify the healing state of grafted seedlings with the high-throughput phenotypic features under the early healing state of melon crops. The N, S-CDs treatment can also promote the growth of roots and scion leaf area during healing. Within 12 days after grafting, the growth of leaf area and root length of the grafted watermelon seedlings treated with carbon point material were significantly greater than those treated without carbon point material, as the time increased. On the 12th day after grafting, the root growth and leaf area in the treatment group were 78.7% and 61.4% higher than those in the control group, respectively. The absorption of nutrients and water was improved for the photosynthesis of plants, the early transplantation of melon grafted seedlings, whereas, the healing time of melon grafted seedlings was shortened with less manpower, material and financial resources. The finding can provide a strong reference to promote the development of grafted seedlings production and seedling industry.