Aims. This study aimed to determine the expression and clinical significance of a cartilage protein, cartilage oligomeric matrix protein (COMP), in knee osteoarthritis (OA) patients. Methods. A total of 270 knee OA patients and 93 healthy controls were recruited. COMP messenger RNA (mRNA) and protein levels in serum, synovial fluid, synovial tissue, and fibroblast-like synoviocytes (FLSs) of knee OA patients were determined using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and immunohistochemistry. Results. COMP protein levels were significantly elevated in serum and synovial fluid of knee OA patients, especially those in the advanced stages of the disease. Serum COMP was significantly correlated with radiological severity as well as measures of body composition, physical performance, knee pain, and disability. Receiver operating characteristic curve analysis unveiled a diagnostic value of serum COMP as a biomarker of knee OA (41.64 ng/ml, area under the curve (AUC) = 1.00), with a sensitivity of 99.6% and a specificity of 100.0%. Further analysis uncovered that COMP mRNA expression was markedly upregulated in the inflamed synovium of knee OA, consistent with immunohistochemical staining revealing localization of COMP protein in the lining and sub-lining layers of knee OA inflamed synovium. Most notably, relative COMP mRNA expression in knee OA synovium was positively associated with its protein levels in serum and synovial fluid of knee OA patients. In human knee OA FLSs activated with tumour necrosis factor-alpha, COMP mRNA expression was considerably up-regulated in a time-dependent manner. Conclusion. All results indicate that COMP might serve as a supportive diagnostic marker for knee OA in conjunction with the standard diagnostic methods. Cite this article: Bone Joint Res 2024;13(6):261–271