Excessive nitrate accumulation in agricultural soils has become a pressing global challenge, negatively impacting crop yield and quality while posing serious environmental risks. Melatonin, a pleiotropic molecule, offers a sustainable solution by concurrently boosting stress resilience and growth regulation in plants, but the molecular mechanisms in alleviating high-nitrogen stress remain unclear. In this study, we optimized melatonin concentrations (10, 50, and 100 µM) through preliminary trials and applied them exogenously via hydroponic root drenching in tobacco (Nicotiana tabacum cv. K326). The result indicated that 50 µM melatonin significantly improved tobacco growth under high nitrate stress (235 mM). Using integrated physiological, biochemical, transcriptomic analysis, and gene functional identification, we elucidated the mechanisms underlying melatonin-mediated stress mitigation in the established "tobacco-high nitrate stress" model. High nitrate stress significantly reduced plant growth, chlorophyll content, photosynthetic efficiency, and nitrogen assimilation while increasing oxidative damage (MDA and H₂O₂). Melatonin application restored growth parameters, enhanced nitrogen metabolism by upregulating key enzymes (NR, NiR, GS, GOGAT, and GDH) and their corresponding genes, and improved photosynthetic performance by stabilizing chlorophyll content and upregulating NtMDH1/2 expression. Transcriptomic analysis revealed melatonin-mediated enrichment of pathways related to nitrogen metabolism and photosynthesis. Furthermore, we found that under high nitrate stress, NtbHLH96 mediated the transcriptional gene expression of nitrogen metabolism after melatonin treatment. Our findings shed light on the molecular basis for the application of melatonin in sustainable agriculture under high nitrate conditions.