Root zone microbial structure is particularly complex in plants with rhizosheaths, and greater understanding of the rhizosheath may play an important role in the future development of sustainable agricultural practices. However, one important reason to focus study on rhizosheath microbial structure is that there is no definite method for rhizosheath separation. The aim of this study was to explore rhizosheath isolation methods and the diversity characteristics of microorganisms around the rhizosphere. In this study, we isolated the rhizosheath of Stipa grandis, a dominant species in desert steppe, and the microorganisms in the roots, root epidermis, rhizosheath and rhizosphere soil were extracted and sequenced by 16S rRNA and ITS. The alpha diversity index of bacteria in Stipa grandis rhizosphere soil was the greatest, followed by rhizosheath, and the alpha diversity index of endophytic bacteria in root system was the smallest. The alpha diversity index of fungi in the rhizosheath and rhizosphere soil were significantly higher than that in the root epidermis and root system. There were significant differences in bacterial community structure between the root epidermis, endophytic bacteria, rhizosheath and rhizosphere soil. Unlike bacterial community structure, the community structure of fungi in the root epidermis was similar that of endophytic fungi, but significantly different from those in rhizosheath and rhizosphere soil. This study demonstrated a feasible method for separating plant rhizosheath and root epidermis. We suggest that the root epidermis can act as the interface between the host plant root and the external soil environment. We will have to re-examine the biological and ecological significance of rhizosheath and microorganisms in rhizosheath, as well as the mechanism explaining the close relationship of the rhizosheath and the plant root epidermis. This study provides theoretical and technical guidance for the isolation of the plant rhizosheath and the study of microorganisms in plant rhizosheath.