Abstract: 【Objective】This study was conducted to clarify the diversity of endophytic flora in roots of Dendrobium nobile （Lindl.） （Orchidaceae） cultivated on two different substrates， so as to provide reference for investigating the influence of differences in endophytic composition on the accumulation of effective medicinal components of D. nobile， as well as optimizing planting conditions.【Method】Two-year D. nobile root samples planted on Danxia stone and volcanic stone substrates were selected for high-throughput sequencing of the V4 region and ITS2 region of 16S rRNA. The secondary metabolites，microbial composition， alpha diversity，beta diversity， and function prediction analysis of endophytic bacteria and endophytic fungi in D. nobile roots were performed.【Result】Dendromoniliside C or isomer（288-p）and Dendromoniliside D or isomer （123-n），two representative sesquiterpene glucosides，were significantly different in roots of D. nobile cultivated at Danxia stone and volcanic stone（P＜0.05， the same below）. Microbial composition analysis showed no significant difference at phylum and genus levels between the two substrate groups. Proteobacteria was the most abundant bacteria at phylum level and Sphingomonas was the most abundant bacteria at genus level. Ascomycota was the absolutely dominant fungal phylum， Phaeosphaeria was the predominant fungal genus. Principal component analysis reflected great differences between volcanic stone and Danxia stone， and the inter-group differences were larger than the intra-group differences based on Unweighted UniFrac index，Bray Curtis index and Jaccard index in ANOSIM test. Furthermore，according to the results of fungal function prediction， in the D. nobile roots of volcanic stone and Danxia stone，the difference of Pathotroph-Saprotroph and Saprotroph among endophytic fungi was the most significant，and there was a significant difference between Fungal Parasite-Plant Pathogen-Plant saprotroph and Undefined Saprotroph. Both were significantly higher in Danxia stone than volcanic stone.【Conclusion】The two substrate planting environments significantly affect the secondary metabolites，relative abundance，dominant microbial community，and community function of the endophytic microbiota in D.nobile roots. At the same time，it also suggests that endophytic fungi in the root system of D. nobile are more sensitive to different planting substrate conditions compared to endophytic bacteria.