Chlorpyrifos is one of the most widely used pesticides that acts on the nervous system by inhibiting acetylcholinesterase. Prolonged use of chlorpyrifos causes severe neurological, autoimmune, and persistent developmental disorders in humans. Therefore, in this study, a highly sensitive and robust biosensor platform was devised by fabricating graphene field effect transistors (graFET) on Si/SiO2 substrate for the detection of chlorpyrifos in real samples. Anti-chlorpyrifos antibodies were immobilized successfully on the graphene surface. Under optimal conditions, graFET sensor showed an excellent response for chlorpyrifos detection in the linear range of 1 fM to 1 µM with a limit of detection up to 1.8 fM in spiked samples. The developed graFET biosensor is highly stable, sensitive, and specific for chlorpyrifos as confirmed by its significant ability to detect changes in electrostatic potential. These findings signify useful efficacy of immunobiosensors for the detection of chlorpyrifos and other organophosphates in fruits and vegetables.