Objective To analyze the epidemiological characteristics of intestinal flora in children with rotavirus enteritis (RVE) in Qinghai, and to provide a basis for clinical treatment and prevention. Methods A total of 64 children with RVE were enrolled in the RVE group, and 62 healthy children who received physical examination in our hospital during the same period were enrolled in the healthy control group. Stool specimens were collected, and fluorescence quantitative PCR technology was used to detect 16S rRNA of feces specimens for intestinal floras including Bifidobacterium, Lactobacillus, Escherichia coli and Enterococcus. The contents of common intestinal flora in two groups were compared. The changes in the contents of four major intestinal bacteria in RVE children of different genders, ages, nationalities and onset seasons were analyzed. Results Compared with the healthy control group, fecal contents of Bifidobacterium and Lactobacillus in the RVE group were significantly reduced (P<0.05). There was a significant difference in the fecal contents of Bifidobacterium and Lactobacillus in RVE children with different ages (P<0.05). The fecal contents of Escherichia coli and Enterococcus in children with RVE from 0 to 1 years old were significantly lower than those in other age groups (P <0.05); (P<0.05). Fecal Bifidobacterium content in RVE children of Han nationality was significantly higher than that of Tibetan children, while Lactobacillus content was significantly lower than that of Tibetan children (P<0.05). The fecal contents of Bifidobacterium and Lactobacillus of RVE children in autumn and winter were significantly lower than those in other seasons (P<0.05). Conclusion In Qinghai, rotavirus enteritis has a great influence on the contents of Bifidobacterium and Lactobacillus in children's intestinal tract. The contents of main intestinal bacteria are different in children with rotavirus enteritis of different ages, nationalities and seasons.