Abstract: The optimization of blanket-seedling culture technology can further integrate tobacco-agronomic characteristics and modern agricultural machinery. To explore the effects of volumetric substrate water content on the growth and root root-knotting force of blanket tobacco seedlings, three different substrate water content treatments were set up in this study for tobacco seedlings from 4 leaves and 1 center to the adult seedling stage, which were 25% (W1), 35% (W2) and 45% (W3), respectively. The results showed that the plant height, shoot biomass, total root length, total root surface area, and total root volume of tobacco blanket seedlings of W2 were better than those of W1 and W3 after 15 days of treatment. However, there was no root-knotting force difference among the three treatments. At 30 days, the root dry mass, total root length, and total root surface area of W1 were significantly higher than those of W2 and W3. And the root-knotting force of W1 and W2 was significantly higher than that of W3, increasing by 21.96% and 15.59%, respectively. In summary, a substrate water content of about 35% for 4 leaves and 1 center to 5 leaves and 1 center (0~15 days after moisture treatment), and about 25% for 5 leaves and 1 center to 7 leaves and 1 center (15~30 days after moisture treatment) is more conducive to the development of tobacco seedlings, including promoting root systems growth and improving root-knotting force.