Background Osteoarthritis is a chronic degenerative disorder with rising prevalence. Early detection of structural damage is difficult. Consequently, there is a pressing demand for reliable biomarkers that enable an earlier diagnosis of osteoarthritis. The aim is to investigate the level of serum fibulin-3 in patients with primary knee osteoarthritis and its correlation with disease severity. Subjects and methods A case–control study was conducted at the Baqubah Teaching Hospital from November 2023 to January 2024. One hundred twenty persons participated in this study (eighty females with the diagnosis of knee osteoarthritis in its early and late stages, and forty age-matched, apparently healthy control. Serum fibulin-3, ESR, CRP, and calcium levels were measured for all participants. Ethical approval was obtained. SPSS was used for data analysis. Results Patients with osteoarthritis had considerably higher serum levels of fibulin-3. In patients with late-stage knee osteoarthritis, this rise was greater than in earlier stages. Serum fibulin and ESR are positively correlated. Fibulin’s area under the curve is 0.830 for diagnosis and 0.709 for differentiating between osteoarthritis’s early and late stages. Conclusion Serum levels of fibulin-3 can act as diagnostic markers for OA and may be useful in determining the severity of knee osteoarthritis.