Field-effect-based capacitive electrolyte–insulator–semiconductor (EIS) sensors have been utilised for the deoxyribonucleic acid (DNA) immobilisation and hybridisation detection as well as for monitoring the layer-by-layer adsorption of polyelectrolytes (anionic poly(sodium 4-styrene sulfonate) (PSS) and cationic poly(allylamine hydrochloride) (PAH)). The EIS sensors with charged macromolecules have been systematically characterised by capacitance–voltage, constant-capacitance, impedance spectroscopy and atomic-force microscopy methods. The effect of the number and polarity of the polyelectrolyte layers on the shift of the capacitance–voltage curves has been investigated. Alternating potential shifts of about 30–90 mV have been observed after the adsorption of each polyanion and polycation layer, respectively. The DNA immobilisation and hybridisation signals were 35–55 and 24–33 mV, respectively. The possible mechanisms for the sensor responses are discussed.