Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease with varying severity, making accurate assessment critical for effective management, Fecal and blood markers have emerged as probable non-invasive tools for evaluating disease activity in UC. Aim: This study aimed to evaluate the effectiveness of combined fecal and blood markers in assessing UC severity. Methods: In a cross-sectional case-control study, 50 UC patients and 25 matched healthy controls were analyzed. Clinical data, along with albumin, complete blood count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), fecal calprotectin, and serum matrix metalloproteinase 9 (MMP-9) levels, were measured. Disease activity was assessed using the Mayo score and correlated with these biomarkers. Results: UC patients had significantly higher levels of ESR, CRP, and fecal calprotectin compared to controls (p < 0.001). MMP-9 levels were also significantly elevated in UC patients (1231.92 ± 612.93 ng/ml) versus controls (653.24 ± 265.17 ng/ml, p < 0.001). MMP-9 showed a positive correlation with bowel movements per day (r = 0.347, p = 0.013), bowel movements per night (r = 0.279, p = 0.050), SCCAI score (r = 0.408, p = 0.003), and CRP level (r = 0.281, p = 0.050). The optimal MMP-9 cut-off to distinguish UC patients from controls was > 471 ng/ml, with 78% sensitivity and 66% specificity. Conclusion: elevated serum MMP-9 and fecal calprotectin levels in UC patients correlate with disease activity and can be useful non-invasive markers for evaluating UC severity.