Abstract: Root cells are important research materials for multi-omics of plant single-cell. As one of the main model species, the efficient isolation of protoplasts from tobacco root cells has becoming increasingly pivotal. To obtain protoplasts from tobacco root cells with high yield and vitality, protoplasts were prepared with enzymolysis method by taking Nicotiana benthamiana and tobacco cv. K326(Nicotiana tabacum)as sample materials. In the meantime, the factors affecting the separation and purification of the protoplasts from root cells, including enzymolysis solution combination, hydrolysis time, osmotic pressure and so on, were optimized. The vitalities of isolated protoplasts were examined under microscope by Trypan blue staining. The results showed that: 1)The protoplast yield and vitality from the root cells were relatively higher after 2 hours of enzymatic hydrolysis with 1.5% cellulase + 1.5% pectinase + 0.4 mol/L mannitol and centrifugation at 500 r/min for 10 min. 2)The obtained tobacco root protoplasts featured high quality with the vitality greater than 70% and reached 10⁵ orders of magnitudes.