Inflammatory Bowel Disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), requires a combination of procedures and tests in diagnosis and discrimination. This study aimed to delineate specific serum metabolomic biomarkers that diagnose IBD and differentiate IBD subgroups. Serum samples and clinical metadata of the participants, IBD patients and Normal Controls (NC), were collected. Untargeted and targeted metabolomic analyses by high-resolution mass spectrometry and multivariate statistical approaches were applied. Further, Receiver Operating Characteristic (ROC) curves, pathways, and network analyses were conducted. Distinct clustering separated IBD patients from the NC, although the CD and UC subgroups overlapped in the non-targeted profiling. Targeted metabolomics revealed elevated tryptophan and indole-3-acetic acid levels and reduced primary-to-secondary bile acid ratios in both CD and UC patients. The differences in specific tryptophan metabolites between CD and UC were identified. The ROC analysis underscored the discriminatory power of the biomarkers (AUC values: NC vs. CD = 0.9738
NC vs. UC = 0.9887
UC vs. CD = 0.7140). Pathway analysis revealed alterations in glycerolipid metabolism, markedly differentiating UC from CD. Network analysis correlated metabolomic markers with the clinical phenotypes of IBD. Serum metabolomic biomarkers can precisely identify IBD, discriminate IBD subtypes, and further reveal the phenotypes of IBD.