Preparations from Echinacea are among the most widely used herbal medicines. Most uses of Echinacea are based on the reported immunological properties. In this paper, we used callus of Echinacea augustifolia for isolation and researched the factors influencing the process of protoplasts preparation, the result indicated it was easy to isolate protoplast from buff-green Callus in E. augustifolia which was looked like granule with symmetrical character. The result showed that the best enzyme solution concentration is composed of cellulase 2.0% (w/v), pectinase1.0% (w/v), hemicellulase 0.5% (w/v), 0.7 mol/L mannitol and 50.0 x 10(4) gFW(-1) protoplasts were obtained after 8 h later. The yield of protoplasts was significantly influenced by cellulase concentration and incubation time. Finally, we summarized an integrated approach including callus induction, callus culture, isolation, purification, identification and calculation of protoplasts.