The invention discloses a glucose detection sensor, and preparation and application methods thereof. The sensor comprises a three-electrode system, wherein a graphene / chitosan / glucose oxidase deposition modified glassy carbon electrode is used as a working electrode, a platinum sheet is used as a counter electrode, and a calomel electrode is used as a reference electrode. In the detection process, by using the graphene / chitosan / glucose oxidase deposition modified glassy carbon electrode as the working electrode, the platinum sheet as the counter electrode and the calomel electrode as the reference electrode, an electrochemical work station is utilized to determine the glucose content. Compared with the traditional method, the invention has the advantages of higher sensitivity and higher specificity, and the low detection limit is 4.0*10<-7> mol / L; and meanwhile, the glucose sensor has the advantages of simple preparation process, stable performance and short sample detection time, can be recycled, and is convenient to operate.