The current study explores potential characteristic metabolic signatures associated with the high cholesterol (CHO), and the progression of coronary artery stenosis (CAS) in high-CHO patients. A metabolomics strategy based on ultra high-performance liquid chromatography/MS-MS and multivariate statistics has been implemented to identify potential biomarkers in high-CHO patients with different levels of CAS. The current study included 57 individuals, comprising 17 healthy paticipants, and 40 high-CHO patients. The high CHO patients were subgrouped based on the computed tomography angiography results, that is, CHO+ no ART (n = 10), CHO+ ART less than 50% (n = 13), CHO+ ART 50-75% (n = 11), and CHO+ ART more than 75% (n = 6). After metabolomics study, 16 discriminating metabolites in positive ion mode and 17 discriminating metabolites in negative ion mode were regarded as possible biomarker candidates to reflect metabolic traits differences between patients with healthy subjects and CHO. A total of six metabolites were tentatively identified as potential biomarkers for the progression diagnosis of CAS: three lysophosphatidylcholines (Lyso-phosphocholine, lysoPC and Lysopersicon esculentum, lysoPE), proline betaine and tryptophan, and prasterone sulfate. The results demonstrated that tryptophan and proline betaine could differentiate the patients with or without high CHO. Tryptophan, prasterone sulfate, LysoPE (0 : 0/18 : 2) or LysoPE (18 : 2/0 : 0), and LysoPE (0 : 0/18 : 1) or LysoPE (18 : 1/0 : 0) could differentiate the patients with severe stenosis (ART > 70%) from the healthy or mild stenosis ones. Proline betaine and significant decrease of LysoPC (17 : 0) could also be a promising biomarker for the mild stenosis (ART