Multiple sclerosis is mediated by the immune system that damages the myelin sheath. Most patients experience inflammation. Since one of the factors that have a role in reducing inflammation is acetylcholine, and according to the benefits of saliva, in this study, the level of salivary and serum cholinesterase activity in patients with multiple sclerosis and healthy were evaluated. Thirty women with multiple sclerosis who were hospitalized in the neurology ward of Imam Reza and Hazrat Rasoul Hospitals and 30 healthy females participated in the study. The severity of multiple sclerosis was calculated by expanded disability status scale (EDSS). Saliva and serum samples were collected in the morning. Cholinesterase activity was assessed by a photometric method. The mean cholinesterase activity in stimulated and unstimulated saliva and serum significantly reduced in the multiple sclerosis group. The cutoff for differentiation of multiple sclerosis patients from healthy individuals by assessing cholinesterase activity (IU/L) was 3577 in serum, 241 in unstimulated saliva, and 266 in stimulated saliva. It seems that cholinesterase activity decreases in patients with multiple sclerosis.