Abstract A label-free and highly sensitive impedimetric aptasensor based on a polyamidoamine dendrimer modified gold electrode was developed for the determination of thrombin. Amino-terminated polyamidoamine dendrimer was firstly covalently attached to the cysteine functionalized gold electrode through glutaraldehyde coupling. Subsequently, the dendrimer was activated with glutaraldehyde, and amino-modified thrombin aptamer probe was immobilized onto the activated dendrimer monolayer film. The layer-by-layer assembly process was traced by surface plasmon resonance and electrochemical impedance spectroscopy. After electrode preparation, the detection of thrombin was investigated in the presence of the reversible [Fe(CN) 6] 3−/4− redox couple using impedance technique. The results showed that the charge-transfer resistance ( R ct) value had a linear relationship with the concentrations of thrombin in the range of 1–50 nM, and the detection limit (S/N = 3) as low as 0.01 nM was obtained. The covalent immobilization of dendrimer on the electrode surface not only improved the immobilization capacity of probe molecules but also magnified the response signal. The aptasensor exhibited favorable regeneration ability, selectivity and stability. It also showed the detectability in biological fluid.