Nanoscale field-effect transistors (FETs) represent a unique platform for real time, label-free transduction of biochemical signals with unprecedented sensitivity and spatiotemporal resolution, yet their translation toward practical biomedical applications remains challenging. Herein, we demonstrate the potential to overcome several key limitations of traditional FET sensors by exploiting bioactive hydrogels as the gate material. Spatially defined photopolymerization is utilized to achieve selective patterning of polyethylene glycol on top of individual graphene FET devices, through which multiple biospecific receptors can be independently encapsulated into the hydrogel gate. The hydrogel-mediated integration of penicillinase was demonstrated to effectively catalyze enzymatic reaction in the confined microenvironment, enabling real time, label-free detection of penicillin down to 0.2 mM. Multiplexed functionalization with penicillinase and acetylcholinesterase has been demonstrated to achieve highly specific sensing. In addition, the microenvironment created by the hydrogel gate has been shown to significantly reduce the nonspecific binding of nontarget molecules to graphene channels as well as preserve the encapsulated enzyme activity for at least one week, in comparison to free enzymes showing significant signal loss within one day. This general approach presents a new biointegration strategy and facilitates multiplex detection of bioanalytes on the same platform, which could underwrite new advances in healthcare research.