We aimed to detect neopterin concentrations in serum and synovial fluid (SF) of knee osteoarthritis (OA) patients and to clarify their relationship with clinical severity of the disease. We cross-sectionally enrolled 176 knee OA patients and 63 age- and sex-matched controls. We measured neopterin concentrations by enzyme-linked immunosorbent assay (ELISA) and investigated the correlation between serum/SF neopterin concentrations and Kellgren-Lawrence (KL) grades as well as Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores in OA patients. Our results demonstrated that increased SF neopterin concentrations were independently correlated with greater symptomatic and radiographic severity in OA patients. These results suggested a crucial role of neopterin activation in the development and progression of knee OA. Assessment of neopterin levels in SF is a potential biomarker to evaluate disease severity in OA patients.