Objective To investigate the effects of mesalazine enteric-coated tablets combined with bifidobacterium tetrad viable tablets on intestinal flora and serum inflammatory mediators in patients with ulcerative colitis (UC). Methods The clinical data of 114 patients with UC admitted to our hospital from January 2022 to July 2023 were retrospectively collected. According to different treatment regimens, the patients were divided into control group (57 cases were treated with bifidobacterium tetrad viable tablets) and observation group (57 cases were treated with mesalazine enteric-coated tablets and bifidobacterium tetrad viable tablets). The clinical efficacy, intestinal flora, intestinal mucosal barrier function (diamine oxidase, D-lactic acid), serum inflammatory mediators [C-C motif chemokine ligand 11 (CCL11), high mobility group protein B1 (HMGB1), MUC1] and adverse reactions of the two groups were analyzed and compared. Results The total effective rate of observation group was significantly higher than that of control group (P < 0.05). Compared with before treatment, the numbers of enterobacter and enterococcus were significantly decreased, and the numbers of lactobacillus and bifidobacterium were significantly increased in both groups after treatment (P < 0.05). The variation range of microflora in observation group was significantly higher than that in control group (P < 0.05). Compared with before treatment, the D-lactic acid and diamine oxidase levels were significantly decreased in both groups after treatment, and the D-lactic acid and diamine oxidase levels in the observation group were significantly lower than those in the control group (P < 0.05). Compared with before treatment, CCL11, HMGB1 and MUC1 in both groups were significantly decreased after treatment, and the reduction range in observation group was significantly greater than that in control group (P < 0.05). There was no significant difference in the incidence of adverse reactions between the two groups (P > 0.05). Conclusion Mesalazine enteric-coated tablets combined with bifidobacterium tetrad viable tablets in the treatment of UC can inhibit inflammation, regulate intestinal flora balance, improve intestinal mucosal barrier function, improve efficacy and high safety.