Avidin-biotin interaction is the strongest known protein-ligand interaction and is widely used in the field of biomedicine, including immunochromatography and various biological detection technologies. Because avidin or streptavidin can specifically bind to four biotinylated molecules efficiently and quickly, and the biotinylated molecules will not change their inherent characteristics. At present, this avidin-biotin interaction has been widely used in the field of biomedical detection to amplify biological signals for specific molecules. Combined with the graphene fieldeffect transistor biosensor, using the avidin-biotin interaction, the concentration of specific molecules in the test solution is expected to be amplified by the biotin-avidin interaction, which helps to increase the limit of detection for the biosensor. Besides, based on the avidin-biotin interaction, the graphene field-effect transistor biosensor is expected to rapidly quantitative detect free biotin and biotinylated molecules. This chapter mainly introduces the graphene field-effect transistor biosensor from the perspectives of device preparation, surface modification, sensitivity, and specificity, and discusses the possible situations that the sensor may encounter in actual detection.Avidin-biotin technology is widely used in different types of ELISA (enzyme-linked immunosorbent assay) kits, polymer-based detection and labeled immunosensor s for the detection of different bio-markers linked to different diseases such as cancer and influenza. In this section, we introduced the employing avidin-biotin technology in the graphene FET biosensor and demonstrated the specific detection of the biotinylated biomolecule in the sub-pico molar (pM) range. The sensing performance of graphene FET biosensor was characterized by the real-time two-terminal electrical current measurement upon injection of analyte solution into a silicone pool preattached onto the graphene channel. Since the Avidin-biotin technology has strong affinity and specificity, any biotinylated biomolecules are hopping for rapid detecting with ultra-low concentration level through this sensing platform. Thus the present graphene FET biosensor is expected to be a breakthrough in biomedical analysis. It can be used as a potential common platform for the rapid and point of care detection of different biomolecules and biomarkers linked to different diseases.