Abstract: 【Objective】 This study aimed to elucidate the mechanisms by which overdominance gene contributed to biomass heterosis in tobacco leaves through transcriptomic analysis，thereby providing theoretical reference for the breeding of high-yield hybrids. 【Method】 The study utilized a hybrid（K326×GDH88）exhibiting biomass heterosis and its parental lines as experimental materials. By measuring the biomass of the tobacco parents and the hybrid，the critical period for heterosis formation was identified. Transcriptome sequencing technology was employed to analyze differentially expressed genes（DEGs）associated with biomass heterosis in tobacco leaves and their expression patterns. 【Result】 Positive heterosis in biomass was observed in the hybrid between 45 and 52 d after transplantation. The mid-parent advantage was the highest between 45 and 52 d post-transplantation. During this period，the gene expression model of the hybrid shifted from an additive mid-parent model to a positive dominance model，indicating that 45 d post-transplantation was the critical period for analyzing biomass formation in hybrid tobacco leaves. Transcriptome sequencing of samples collected 45 d after transplantation revealed 2660 DEGs between the hybrid and its parents. Of these，1683 DEGs（63.27%） exhibited overdominant expression，which was likely the primary cause of biomass heterosis in tobacco leaves. Among the overdominantly expressed DEGs，28 were up-regulated and enriched in photosynthesis-related pathways，while 10 were down-regulated and enriched in sucrose and starch metabolism pathway related to respiration. These DEGs with overdominant expression in photosynthesis and respiration were potential key candidate genes regulating biomass heterosis in hybrid. 【Conclusion】 The critical period for the formation of biomass heterosis in tobacco hybrid leaves is 45 d posttransplantation. The formation of biomass heterosis is primarily influenced by the overdominance effect of DEGs. The overdominant up-regulation and down-regulation of related DEGs in photosynthesis and respiration may be the underlying reasons for biomass heterosis in tobacco leaves.