The factors effecting isolation and culture of apple protoplast from leaves in vitro were studied. The results indicated that the optimum enzyme solution for apple leaves was Cellulase-Onzuka R-10 0.8% + Pectinase 0.5% + PVP 1% + mannitol 0.65 mol/L + MES 0.1%. The appropriate culture density of protoplasts was 1×105 (No. of protoplasts/ml) and the proper culture method was Liquid-Solid-Double-Layer Culture. The protoplasts were cultured on the medium of modified MT + BA 1.0 mg/L + 2.4-D 0.2 mg/L + mannitol 6.5 mol/L + Vc 5 mg/L + Glu 500 mg/L + CH 100 mg/L + ME 500 mg/L + Arg 50 mg/L. Cell enlargement was found in 1-2 days and cell division was found in 3-4 days. Cell division took place 3-5 times after 2 weeks culture. Cell colony was found in one month and microcalli in two and half months of Malus hupehensis Rehd. The cell number of the cell colony of M7 and Lujia 5 were 7 to 10. And no cell division was found in Gala.