Rheum palmatum L. (Chinese rhubarb) is a highly regarded medicinal plant. Its dominant active constituents are anthraquinones including emodin, aloe-emodin, rhein, etc. Rhein naturally occurs in anthraquinone (1, 3, 8-trihydroxy-6-methyl anthraquinone), which is found in R. palmatum L. and related plants such as rhubarb. It has good antitumor, anti-inflammatory, anticancer, antimicrobial and hemostatic properties. In this study, a total of 14 strains of endophytic fungi were isolated from R. palmatum L. All fungal isolates were fermented in liquid PDA medium and their extracts were preliminarily analyzed by antibacterial reactions, magnesium acetate-methanol reagent and Borntraiger's reaction, and the strain reselection was made by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and LC-MS to identify the fermentation products of the selected strains and confirmed through a comparison with authentic standards. Extract from one strain, R13, showed positive reactions with both reagents. The strain R13 had a component with the same TLC (Rf) value and HPLC, LC-MS retention time as authentic rhein standards. The yield of rhein in R13 can reach 5.672mgl(-1). The fungi were identified as Fusarium solani by using both ITS rDNA sequencing and spore morphology.