We investigated the expression of miRNAs as a potential biomarker in the colon, rectum, plasma, and feces of the rat dextran sulfate sodium (DSS)-colitis model. A 5% DSS solution with drinking water was administered to male SD rats for 1 week, followed by a 1-week off-dose period. In-life parameters were examined daily, and colon length and pathological changes were assessed post-mortem. A selected panel of miRNAs (miR-16-3p, miR-21-5p, miR-31a-5p, miR-34a-5p, miR-146b-5p, miR-155-5p, miR-181b-5p, miR-221-5p, and miR-223-3p) was also measured in the colon, rectum, plasma, and feces using digital polymerase chain reaction (PCR). A high disease activity index (DAI) and reduction in colon length were observed in DSS-treated rats. Erosion and inflammatory cell infiltration were evident in the colon and rectum after DSS treatment. These parameters tended to recover, and regenerative hyperplasia was observed in the rectum after the recovery period. After the end of the administration period, all nine selected miRNA levels showed lower expression in colonic and rectal tissues. miRNA levels, except miR-21-5p and miR-155-5p, were lower in both plasma and feces at 5% DSS group. In contrast, at the end of the recovery period, miRNA levels tended to increase in all samples. Particularly, a higher expression of miR-31a-5p, miR-181b-5p, and miR-223-3p was observed in feces. We suggest that the miRNAs from colon, rectum, plasma, and feces are potential quantitative and sensitive biomarkers in the rat DSS-colitis model. In particular, miR-31a-5p, miR-181b-5p, and miR-223-3p from feces could be used as non-invasive biomarkers to evaluate the reversibility in DSS-treated rats.