Abstract: In the present study, the effect of mannitol concentration, combination of different enzyme (cellulase and macerozyme) solutions, and enzymolysis time on the separation of mesophyll protoplasts and purification of protoplasts from two high density separating solutions were studied in the leaves of tea seedlings. The results demonstrated that high quality mesophyll protoplasts were obtained from tea leaves subjected to enzymatic digestion in a solution containing 0.6 mol/L mannitol, 1.5% cellulase, and 2.5% macerozyme for 16-18 h in the dark. In addition, protoplasts gathered into a band on the layer between 10% mannitol and 25% iodixanol.