Abstract: To analyze the formation mechanism of biomass heterosis in tobacco leaves from the perspective of photosynthetic physiology, this study used six parents and their hybrid combinations with significant biomass differences to measure biomass, photosynthetic characteristics, chlorophyll contents, and expression levels of photosynthetic-related genes in the tested materials. The relationship between biomass heterosis in tobacco leaves and photosynthetic characteristics of hybrids as well as heterosis formation mechanism were analyzed based on the photosynthetic characteristics of these hybrid species. Results showed that the biomass of tobacco hybrids had heterosis, with high-parent heterosis and mid-parent heterosis rate of 55.56% and 77.78%. In addition, Photosynthetic related indicators such as net photosynthetic rate, intercellular CO₂ concentration, and chlorophyll a content of the hybrid showed heterosis, with the combination rates of high-parent heterosis and mid-parent heterosis being 55.56%, 100%, 66.67% and 88.89%, 100%, 77.78%, respectively. There is a significant correlation between biomass heterosis and net photosynthetic rate, with correlation coefficients of 0.73 (p<0.05). The relative expression levels of NtLhcb1, NtLhcb2, NtPsbA, NtatpF, and Ntrbcl genes related to photosynthesis showed significant differences between strong and weak heterosis hybrids, and their expression levels were significantly positively correlated with the heterosis of biomass. The results indicate that the up-regulation of NtLhcb1, NtLhcb2, NtPsbA, NtatpF, and Ntrbcl genes in the hybrids enhances the photosynthetic capacity of the hybrid.