The technique for mesophyll protoplasts isolation and purification of a triploid poplar variety,Populus alba×P.berolinensis Yinzhong,was studied,for further genetic improvement through cell fusion and transgenic engineering.The leaves of aseptic seedling were used for mesophyll protoplast isolation and purification.The results showed that the yield and viability of the protoplasts were significantly influenced by the concentration of enzyme mixture,osmotic pressure and incubation time.Under the optimum condition for mesophyll protoplast isolation,CPW+3% Cellulase RS+0.5% Macerozyme R-10+0.3% Pectinse Y-23+0.6 mol/L mannitol+0.6 g/L MES+1 g/L BAS with 8 h incubation in dark,the protoplast yield could reach up to 2.13×10⁷ per gram of leaves and the viability could be 80.18%.The best purification method was rising with sucrose density centrifugation.The yields were up to 1.06×10⁷ protoplasts when purified with sucrose at the concentration of 40%.The protocol could meet the demands of protoplast culture-based techniques.