Mann–Whitney U-test showed that there were higher serum fibulin-3 concentrations in the case group compared with the controls. Higher serum and synovial fluid fibulin-3 concentrations were found in knee osteoarthritis subjects compared with those with Kellgren–Lawrence grades 2 and 3. Knee osteoarthritis subjects with Kellgren–Lawrence grade 3 had higher serum and synovial fluid fibulin-3 concentrations compared with those with Kellgren–Lawrence grade 2. Serum and synovial fluid fibulin-3 concentrations were significantly correlated with Kellgren–Lawrence grading after Pearson correlation analysis (r = 0.532, P < 0.001 and r = 0.613, P < 0.001). Multinomial logistic regression analysis demonstrated a significant association between serum and synovial fluid fibulin-3 concentrations with Kellgren–Lawrence grades (P < 0.001 and P < 0.001).