The suspension cells and leaves of Liriodendron tulipifera were used as experiment materials to isolate protoplasts.The influences of different factors including pretreatment osmotic pressure,the kind and combination of enzymes,digestion time,culture medium were compared.The result showed that the pretreatment of leaves and suspension callus cells in the Cell Protoplast Wash containing 0.1% 2-(4-Morpholino) ethanesulfonic acid (MES) and 0.6mol/L mannitol (60M-CPW) for 1 h at 25 ℃ and light-resistant has the best effect in the protoplasts isolation.The best enzyme solution of suspension cells contained 60M-CPW+1% cellulase+1% hemicellulase+0.2% pectinase Y-23+0.1% MES,incubated at 25 ℃ for 6 h and the protoplasts yield was about 3×10⁶ per gram.The best enzyme solution of leaves contained 60M-CPW+2% cellulase+1% hemicellulase+0.2% pectinase Y-23+0.1% MES,incubated at 25 ℃ for 10 h and the protoplasts yield was about 11×10⁶ per gram.It was easy to culture the protoplasts of suspension callus cells and the culture KM8p was better than the modified MS.In the culture medium of KM8p containing 1.0 mg/L 2,4-D+0.5 mg/L 6-BA,the protoplasts of suspension cells will be formed visible callus in 25 days.