This work was aimed to develop reusable magnetite chitosan composite containing carbon paste electrode for biosensor application. Glucose oxidase (GOx) enzyme was used to prepare GOx-magnetite-chitosan nanocomposite containing carbon paste electrode for sensitive detection of glucose. The immobilized enzyme retained its bioactivity, exhibited a surface confined reversible electron transfer reaction, and had good stability. The surface parameters like surface coverage (tau), Diffusion coefficient (D0), and rate constant (kS) were studied. The carbon paste modified electrode virtually eliminated the interference during the detection of glucose. The excellent performance of the biosensor is attributed to large surface-to-volume ratio, high conductivity and good biocompatibility of chitosan, which enhances the enzyme absorption and promotes electron transfer between redox enzymes and the surface of electrode. The shelf life of the developed electrode system is about 12 weeks under refrigerated conditions. We report for the first time in the fabrication of carbon paste bioelectrode containing magnetite-chitosan-GOx.