Objective To compare and analyze the differences in the gut microbiota and serum biochemical markers between the Tibetan people in Hongyuan, and Han people in Chengdu, Sichuan Province with different body mass indexes (BMI). Methods From May 2018 to September 2019, Tibetan and Han residents from Aba Hongyuan area and Chengdu Qingbaijiang area were recruited based on the China multi-ethnic cohort (CMEC). Their demographic information, height, body mass, stool and blood samples were collected. According to BMI and ethnic groups, the subjects were subsequently divided into obese and non-obese Tibetans, as well as corresponding Han groups. The intestinal microflora and blood biochemical markers were measured, and the differences among groups were compared and analyzed. Results The triacylglycerol (TG) level in Tibetan population was inferior to that in Han generally. The levels of total liver cholesterol (CHOL), TG, low-density lipoprotein cholesterol (LDL-C), gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were higher, while the level of high-density lipoprotein cholesterol (HDL-C) was lower in the obese population than the non-obese population. The species richness and diversity of gut microbiota were greater in Tibetans than in Han population. The abundance of fusobacterium in the obese Han group was more than that in the non-obese Han (P < 0.05), whereas that of firmicutes showed the opposite (P < 0.05). The abundance of verrucomicrobia in the obese Tibetans was lower than that in the non-obese Tibetans (P < 0.05). At the genus level, the Tibetan group had 12 genera of higher abundance than Han population (P < 0.05), while the abundance of 6 genera in the Han group was greater than that in Tibetan population (P < 0.05). Among them, collinsella genus with superior abundance in the Tibetan population was positively correlated with TG, LDL-C, GGT, ALT, and aspartate aminotransferase (AST), and negatively correlated with HDL-C (|r|>0, P < 0.05). The fusobacterium genus, which was more abundant in Han population, was positively correlated with TG and GGT, and negatively correlated with HDL-C (|r|>0, P < 0.05). Conclusion The gut microbiota structure and biochemical indexes vary considerably among Tibetan and Han population with different BMI. collinsella and fusobacterium may be related to the obesity of Tibetans and Han population, respectively.