Abstract A novel simple protocol of synthesizing tryptamine-functionalized reduced graphene oxide (TRA–rGO) was proposed to fabricate label-free electrochemical impedance DNA biosensor. TRA was anchored to rGO with its indole ring via π-stacking interaction. The morphology, conductivity and interaction of TRA–rGO were characterized by atomic force microscopy, high resolution transmission electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy (EIS) as well as Raman and fluorescence spectroscopy. The amino-substituted oligonucleotide probe was conjugated to TRA by cross-linker glutaraldehyde for preparing an electrochemical biosensing platform. The DNA hybridization reaction of oligonucleotide probe with target DNA was monitored by EIS. Under optimum conditions, the proposed biosensor exhibited high sensitivity and low detection limit for detecting complementary oligonucleotide. The target oligonucleotide could be quantified in a wide range of 1.0×10−12–1.0×10−7M with low detection limit of 5.2×10−13M (S/N=3).