As we are all aware the hormone estrogen is the most essential sex hormone of the female. It plays significant roles in all phases of the life of females. Osteoarthritis is the most debilitating bone disorder commonly seen in females more than males. Out of all the joints knee joint is most commonly affected as it is the weight-bearing joint. So here in this study, the main objective is to know the role of serum estrogen and Nitric oxide levels in post-menopausal knee osteoarthritic female patients. The samples were collected and the chemiluminescence and ELISA method performed the tests. Consequently, results showed decreased levels of serum Estrogen in the study group than in the control group and levels of serum nitric oxide was increased as it confirmed the inflammatory marker at the site of infection. This whole suggests that deficiency estrogen and raised nitric oxide are helpful in the diagnosis and treatment modalities of knee osteoarthritis in postmenopausal females.