The excellent electronic properties of single-layer graphene provide the fundamental basis for its use in advanced electronics applications. Graphene-based biosensors, however, are one of the exceptions among the applications that take advantage of the unique properties of this material, because high hydrophobicity of graphene hinders the controlled immobilization of biomolecular probes, particularly of antibodies for immunoassays. In contrast to methods that rely on aggressive chemical oxidation of graphene, we overcome this challenge by implementing an effective strategy that modifies single-layer graphene to render it hydrophilic and biocompatible without compromising the electronic structure. Graphene produced in-house by chemical vapor deposition (CVD) was modified with a heterobifunctional linker that attached to the surface via a pyrene group and presented a standard N-hydroxysuccinimide (NHS) ester ligand for covalent immobilization of biomolecules. The suitability of thus modified surface for biofunctionalization was then confirmed via real-time monitoring of the immobilization of an antibody by quartz crystal microbalance (QCM) measurements followed by the chemical characterization by x-ray photoelectron spectroscopy (XPS). Finally, as a proof-of-concept for biofunctionalization of graphene without compromising its electronic structure, a graphene immuno-field-effect transistor (immuno-FET) was fabricated, biofunctionalized, and tested for detection of Matrix Metalloproteinase 9 (MMP-9), a biomarker related to clinical diagnostics of ischemic stroke.