Root-knot nematode disease occurs frequently due to continuous mono-cropping and excessive water and nitrogen input. The disease degree and gall index are often used to evaluate the damage of root-knot disease. However, the weak correlation between these two indicators to tobacco leaf dry weight has often been reported. The objective of this study is to verify whether root-knot density (RKD) as a new indicator, the root-knot number per unit root weight or volume, can describe the damage of root-knot disease on tobacco growth and yield quantitatively. A total of 3000 tobacco plants from 60 independent plots were classified according to the damage symptoms of leaves in situ. A total of six plants in each plot were selected and sampled to represent 6 damage levels with a total of 360 plants. The responding roots were taken out with a root auger. Dry weights of leaf, stem, root and root-knot as well as root volume, root-knot number, disease degree and gall index were determined for all 360 plants separately. Our results showed that (1) the disease degree and gall index of root-knot nematodes had a weak negative correlation with tobacco leaf dry weight. Meanwhile, leaf dry weight and the dry weight, volume and number of root-knots were not correlated. (2) The root dry weight, volume and length of diameter ≥ 2 mm roots were significantly positively correlated with leaf dry weight. (3) The RKD of root diameter ≥ 2 mm roots was significantly negatively correlated with the leaf dry weight. (4) The dry weight of leaves, stems and roots decreased significantly with the increase in the average RKD of diameter ≥ 2 mm roots in reclassified groups, which was significantly positively correlated with the average reclassified disease degree and gall index. Our results highlighted that the proposed RKD in this paper can be used to evaluate the damage degree of root-knot disease quantitatively, as a new indicator for future research and practical diagnosis of root-knot nematodes.