Objective: To study the correlation of intestinal flora levels with cytokine and Toll-like receptors (TLRs) molecule expression in patients with ulcerative colitis (UC). Methods: Patients diagnosed with UC in our hospital between May 2013 and August 2016 were selected as UC group (n=57), and the feces samples as well as diseased intestinal mucosa tissue and normal intestinal mucosa tissue were collected; the healthy subjects during the same period were selected as the control group (n=80), and the feces samples were collected. The intestinal flora levels as well as the expression of cytokines and TLRs molecules in intestinal mucosa tissue were detected. Results: Bifidobacterium and lactobacillus levels in intestinal tract of UC group were significantly lower than those of control group (P<0.05) while E.coli, bacteroides and enterococcus levels were significantly higher than those of control group (P<0.05); TLR2, TLR4, TLR5 and TLR9 protein expression in diseased intestinal mucosa were significantly higher than those in normal intestinal mucosa (P<0.05), negatively correlated with the bifidobacterium and lactobacillus levels, and positively correlated with E.coli, bacteroides and enterococcus levels; IL-17, IL-23, HMGB1, Fstl1 and TNF-α protein expression in diseased intestinal mucosa were significantly higher than those in normal intestinal mucosa (P<0.05) and positively correlated with TLR2, TLR4, TLR5 and TLR9 protein expression in diseased intestinal mucosa. Conclusions: Intestinal flora disorders in patients with ulcerative colitis can increase the expression of TLR to promote inflammatory cytokine secretion and activate intestinal mucosal inflammation.