Ulcerative colitis (UC) is a chronic, regressive nature disease. The use of conventional diagnostic procedures such as magnetic resonance imaging, ultrasonography, and X-rays during the dormant and early stages of the disease does not aid in diagnosing the disease. As a result, a novel design, such as labelled compounds, was used to image ulcerative colitis disease. In this study, olsalazine was labeled with [125/131I] and the labelling parameters were adjusted to obtain a high radiochemical yield (98.5%). In addition, the olsalazine radiotracer gave 96.0% purity in rate serum for up to twelve hours before it started to degrade at twenty-four hours, it was stable also, in saline for up to twenty-four hours. Molecular docking was used to assess a complex's affinity for its biological target, and the PPAR receptor. We also performed the biological assessment in mice models of both standard and ulcerative colitis. Results demonstrated that [125/131I] iodoolsalazine had a high uptake of 79.5% (ID/g) at 120 minutes post-injection and still high to 77.00% at 24 hours. So, the labelled compound, [131I] iodoolsalazine, could be considered a new potential selective radiotracer for preclinical diagnostic research of ulcerative colitis.