Mesalazine (MSZ), a first-line treatment for ulcerative colitis (UC), was formulated into acid-resistant, colon-targeted gel microspheres to reduce upper gastrointestinal tract (GIT) exposure and extend drug retention in the colon. In this study, we used MSZ/hydroxypropyl-β-cyclodextrin (MSZ/HP-β-CD) as the model drug, dopamine-modified sodium alginate (DA-SA) and konjac glucomannan (KGM) as the carrier matrix, and chitosan (CS) as the coating material. The colon-targeted gel microspheres (MSZ/HP-β-CD/DA-SA/KGM/CS) were prepared using the drop method. These microspheres had a drug loading capacity of 7.9 ± 0.01 % and an encapsulation efficiency of 72.5 ± 0.03 %. The drug primarily released in the colon environment, showing pH and β-mannanase sensitivity. The dried microspheres measured approximately 0.6 mm, suitable for oral administration. In the rat UC model, after oral administration of gel microspheres, the colon length increased, while the DAI score, spleen index, and the expression levels of IL-6, IL-1β, TNF-α, TLR4, MyD88 and NF-κB p65 all decreased. Histopathological examination showed that treated UC rats' colon tissues closely resembled those of healthy controls. These findings indicate that pH-enzyme-responsive coated gel microspheres can effectively target the colon and show potential for UC treatment.