Abstract: To delve into the intricate mechanisms governing the regulation of early seedling development in tobacco in response to light, the tobacco variety CV87 was chosen as the experimental material. Two treatments were implemented: a 12-hour light period and a continuous darkness, allowing for a comprehensive phenotypic study of morphological changes during the first nine days of tobacco seed incubation. Building upon this foundation, a transcriptomic analysis was employed specifically on the fourth day of development to investigate differential gene expression networks. The findings revealed that seed germination vigor and germination rate showed an increase of approximately 30% under light conditions. Furthermore, the elongation of cotyledon opening increased by approximately 2 cm, while the length of the hypocotyl decreased by approximately 2 cm. During the early stages of seedling development, 551 genes exhibited differential expression induced by light, with 364 genes being upregulated and 187 genes being downregulated. Among these 551 genes, 28 of them were found to interact at the protein level, including upregulated genes such as AMT1-3, PSAK, and LHCB5, as well as downregulated genes such as OLEO1, LEA7, and PDF2.3. Gene Ontology (GO) enrichment analysis demonstrated that these 28 differentially expressed genes were primarily associated with photosynthesis, light systems, and plant hormone responses. In conclusion, this study elucidates the phenotypic variations during early tobacco seedling development in response to light signals. Through transcriptomic analysis, several regulatory genes associated with the observed phenotypic differences were uncovered. These findings serve as a foundation for further research on the impact of light environment on tobacco germination and early seedling development.