The present invention pertains to the field of biotechnology, which discloses a preparation method of streptomyces total protein. The method for separation and preparation of the streptomyces total protein of the present invention is simple and easy to be operated, the separation effect of the streptomyces protein samples is good by using the method, so the expressed total protein of the cells can be obtained as completely as possible, and the obvious protein degradation phenomenon can not appear during the preparation process. The streptomyces total protein which is prepared by the present invention can obtain a two-dimensional electrophoresis pattern with high quality and good reproducibility, so the present invention can facilitate the follow-up bio-mass spectrometry analysis and have greatly practical application value.