Nanomaterials have been extensively utilized in biosensing systems for highly sensitive and selective detection of a variety of biotargets. In this work, a facile, label-free, and ultrasensitive electrochemical DNA biosensor has been developed, based on "urchinlike"carbon nanotube-gold nanoparticle (CNT-AuNP) nanoclusters, for signal amplification. Specifically, electrochemical polymerization of dopamine (DA) was employed to modify a gold electrode for immobilization of DNA probes through the Schiff base reaction. Upon sensing the target nucleic acid, the dual-DNA (reporter and linker) functionalized AuNPs were introduced into the sensing system via DNA hybridization. Afterward, the end-modified single-wall carbon nanotubes with DNA (SWCNT-DNA) were attached to the surface of the AuNPs through linker-DNA hybridization that formed 3D radial nanoclusters, which generated a remarkable electrochemical response. Because of the larger contact surface area and super electronic conductivity of CNT-AuNP clusters, this novel designed 3D radial nanostructure exhibits an ultrasensitive detection of DNA, with a detection limit of 5.2 fM (a linear range of from 0.1 pM to 10 nM), as well as a high selectivity that discriminates single-mismatched DNA from fully matched target DNA under optimal conditions. This biosensor, which combines the synergistic properties of both CNTs and AuNPs, represents a promising signal amplification strategy for achieving a sensitive biosensor for DNA detection and diagnostic applications.