Capacitive field-effect electrolyte-diamond-insulator-semiconductor (EDIS) structures with O-terminated nanocrystalline diamond (NCD) as sensitive gate material have been realized and investigated for the detection of pH, penicillin concentration, and layer-by-layer adsorption of polyelectrolytes. The surface oxidizing procedure of NCD thin films as well as the seeding and NCD growth process on a Si-SiO2 substrate have been improved to provide high pH-sensitive, non-porous thin films without damage of the underlying SiO2 layer and with a high coverage of O-terminated sites. The NCD surface topography, roughness, and coverage of the surface groups have been characterized by SEM, AFM and XPS methods. The EDIS sensors with O-terminated NCD film treated in oxidizing boiling mixture for 45 min show a pH sensitivity of about 50 mV/pH. The pH-sensitive properties of the NCD have been used to develop an EDIS-based penicillin biosensor with high sensitivity (65-70 mV/decade in the concentration range of 0.25-2.5 mM penicillin G) and low detection limit (5 μM). The results of label-free electrical detection of layer-by-layer adsorption of charged polyelectrolytes are presented, too.