Abstract: The large-scale preparation of protoplasts is the key to study the techniques of protoplast transformation, mutation and fusion. The preparation of vacuoles is of great significance for studying the characteristics of decomposition and transport of organic matter in vacuoles. However, the current separation technology is not mature enough. In this study, protoplasts and vacuoles were isolated from the mycelia of Aspergillus sydowii and the separation conditions were optimized. The effect of different factors on the preparation and separation efficiency of protoplasts and vacuoles were expored by using the mycelium of Aspergillus sydowii DJ515-2 as the studying material. The results showed that the maximum protoplast yield from mycelia was 5.167 ×105/m L when the mycelia cultured for 42 h and digested at 25 ℃ for 4 h with the mixed enzyme solution consisting of 3% cellulase, 1%snailase and 3% lysing enzyme. At the same time, on the above basis, the optimal conditions for lysing vacuoles were pH 7.5, 1.0 mol/L KCl and 0.020% of the Triton X-10 content. The maximum vacuole yield was 2.63×105/m L,which was 50.8% of the protoplasts yield. The yield increased by 40%～45% compared with the method of polybasic compound inducing fungal protoplast lysis to release vacuoles. This study could provide a material basis for various protoplast techniques and subcellular-based studies of fungi and plants.