Organophosphate (OP) is a compound considered the main leading cause of morbidity and mortality from poisoning worldwide. Serum pseudocholinesterase was evaluated as a diagnostic indicator; it cannot be used to monitor therapy or severity of the intoxication. The rationale of the current study was to evaluate sensitivity, specificity, and cut-off values of serum S100B and amyloid β for neurological affection severity. This study was carried out on sixty OP-impaired patients; in addition, 20 normal controls were included. Serum liver and kidney function tests, malondialdehyde, pseudocholinesterase, and the levels of S100B and amyloid β (Aβ) were determined. Data showed that Pearson’s analysis indicated that the serum level of S100B was positively correlated with Aβ. On the contrary, the activity of pseudocholinesterase was negatively correlated with both of S100B and Aβ. Serum ALT, AST, creatinine, urea, acetylcholine, and MDA levels were elevated while pseudocholinesterase activity was reduced in moderate and severe OP intoxication versus control. A drastic elevation (p<0.001) in the levels of S100B and Aβ was performed in the patient group suffering from OP intoxication versus the normal group. The diagnostic statistical validation of targeted parameters in distinguishing between moderate OP intoxication and control clarifies that S100B displayed the best AUC (0.997) followed by Aβ (AUC=0.992), while the diagnostic veracity of S100B and Aβ in setting apart severe OP-intoxicated and normal subjects stated the symmetric efficacy of potential markers. It was concluded that the significant changes in the levels of S100B and Aβ were directly proportional to the degree of severity of OP intoxication.