Abstract A penicillin-sensitive potentiometric biosensor with a high long-term stability was fabricated by immobilizing penicillinase on top of a pH-sensitive electrolyte/insulator/semiconductor (EIS) layer structure made of Al/p-Si/SiO 2/Si 3N 4. The immobilization was performed in two distinct steps with a heterobifunctional cross-linker. In the first step the amine-specific functional group of the cross-linker molecules was linked directly to the Si 3N 4 surface. Subsequently, the second reactive group of the fixed cross-linker molecules was made to bind to the enzyme. This immobilization procedure avoids a reduction of activity due to intermolecular cross-linking of the enzyme and results in sensor devices with a high long-term stability of at least 250 days. Sensitivities of about 60 mV per decade of penicillin concentration were obtained from C/V (capacitance/voltage) and CONCAP (constant capacitance) measurements.