Biosensors based on graphene field effect transistors (GFETs) decorated with antibody-functionalized platinum nanoparticles (PtNPs) are developed for the quantitative detection of breast cancer biomarker HER3. High-quality chemical vapor deposited graphene is prepared and transferred over gold electrodes microfabricated on an SiO2/Si wafer to yield an array of 52 GFET devices. The GFETs are modified with PtNPs to obtain a hybrid nanostructure suitable for attachment of HER3-specific, genetically engineered thiol-containing single-chain variable fragment antibodies (scFv) to realize a biosensor for HER3. Physical and electrical characterization of Bio-GFET devices is carried out by electron microscopy, atomic force microscopy, Raman spectroscopy, and current–gate voltage measurements. A concentration-dependent response of the biosensor to HER3 antigen is found in the range 300 fg mL−1 to 300 ng mL−1 and is in quantitative agreement with a model based on the Hill–Langmuir equation of equilibrium thermodynamics. Based on the dose–response data, the dissociation constant is estimated to be 800 pg mL−1, indicating that the high affinity of the scFv antibody is maintained after immobilization. The limit of detection is 300 fg mL−1, showing the potential for PtNP/G-FETs to be used in label-free biological sensors.