<p>Distinguishing intestinal inflammation from colorectal cancer remains challenging using non-invasive biomarkers. tRNA-derived fragments have been implicated in cancer, but their role in Crohn’s disease activity and their ability to differentiate inflammatory from malignant intestinal conditions remain unclear. This study evaluated circulating tRF-Gly-GCC in patients with active Crohn’s disease (<i>n</i> = 20), inactive Crohn’s disease (<i>n</i> = 20), colorectal cancer (<i>n</i> = 24), and healthy controls (<i>n</i> = 21). Serum tRF-Gly-GCC expression levels showed a directional increase across disease states, from controls to inactive Crohn’s disease, active disease, and colorectal cancer. A Jonckheere–Terpstra test formally confirmed a significant monotonic increase (z = 7.896, <i>p</i> &lt; 0.001). The marker distinguished colorectal cancer from controls with high sensitivity (95.8%) and specificity (95.2%), and differentiated colorectal cancer from Crohn’s disease with sensitivity of 95.8% and specificity of 80.0%. It also distinguished active from inactive Crohn’s disease with 90% sensitivity and 90% specificity. In Crohn’s disease, tRF-Gly-GCC correlated with disease activity indices but not with fecal calprotectin. These findings suggest that circulating tRF-Gly-GCC reflects intestinal inflammatory activity and may serve as a non-invasive biomarker for disease assessment and differentiation between inflammatory and malignant conditions.</p>