The rising incidence of ulcerative colitis (UC) in China highlights the need for effective biomarkers. Autoantibodies targeting integrin αvβ6 show association with UC. This study developed a novel chemiluminescent immunoassay (CLIA) for detecting anti-αvβ6 autoantibodies by conjugating recombinant αvβ6 protein to superparamagnetic microparticles and detecting captured IgG with acridinium ester-labeled anti-human IgG. The assay was rigorously validated using 961 serum samples from China. Cutoff values were established using 186 UC patients and 244 healthy controls, while clinical sensitivity was assessed on 531 samples. The assay achieved a sensitivity of 72.7% and specificity of 94.7% (AUC = 0.846) for UC diagnosis, demonstrating exceptional specificity: 100% in children (n = 128) and pregnant women (n = 171), 99.36% in adults (n = 132), and 99.00% in the elderly (n = 100), yielding an overall specificity of 99.35%. These results confirm the high sensitivity and excellent specificity of this CLIA for UC detection, aligning with global findings. Although further research into antibody titer correlations with disease severity is warranted, this assay demonstrates significant potential for clinical application as an auxiliary diagnostic tool for UC.