Dendrobium officinale is widely used as a health supplement, but its specific impact on healthy gut microbiota has not yet been clarified, nor has its impact on different human genders. To overcome the problems mentioned above. DOP was extracted and purified with an 8000–12,000 Da dialysis bag. The molecular weight and monosaccharide composition were determined using HPGPC and GC. Gas chromatography was used to detect the content of SCFA. 16S rDNA sequencing was used to analyze the diversity of human microbiota. The results showed that DOP contained two fractions, with an average molecular weight of 277 kDa and 1318 Da, and mainly composed of mannose and glucose. DOP can increase the relative abundance of benign microbiota and decrease the harmful types. Propionic acid content in women was significantly increased after DOP treatment. Finally, the correlation analysis revealed that DOP was beneficial to the microbiota of both men and women. It can be concluded from the results that DOP is a health supplement suitable for humans, and especially women.