Abstract: Objective To determine the structure and characteristics of intestinal flora in EV71 hand-foot-mouth disease. Methods Fecal samples were collected from healthy children and child patients with EV71 hand-foot-mouth disease who were diagnosed in Suqian Hospital Affiliated to Xuzhou Medical University from January 2020 to December 2021. After DNA extraction, 16S rDNA amplicon sequencing was performed, and the obtained biological information was classified, quantified and compared. Alpha-diversity was used to analyze the differences in abundance and diversity between the two groups. Beta-diversity was used to analyze the differences in bacterial community structure between the two groups. LDA analysis and LEfSe analysis were used to compare the significantly different genera between the two groups. Results A total of 80 fecal samples were collected from children with EV71 hand-foot-mouth disease (the EV71 group, n=45) and healthy children (the control group, n=35). Compared with the control group, there were no significant changes in the diversity (shannon index, P>0.05) and abundance (ace index, P>0.05) of intestinal flora in the EV71 group. However, the structure of intestinal flora in EV71 group was significantly changed (weighted_unifrac, P<0.05), which was characterized in the increased abundance of Neisseria (P<0.05) and Klebsiella (P<0.01), and the deceased abundance of Romboutsia (P<0.01), Dialister (P<0.01) and Clostridium (P<0.05). Conclusions The intestinal flora of EV71 hand-foot-mouth disease is disordered, which is mainly characterized by the decrease of butyrate producing bacteria and the increase of opportunistic pathogenic bacteria. The results provide some theoretical evidence for the pathogenesis and microecological treatment of EV71 hand-foot-mouth disease in the advanced stage.