Vertically aligned diamond nanowires are biofunctionalized using aminophenyl linker molecules to bond nucleic acid molecules with a well-defined nanometer-sized spacing to the transducer. This novel DNA biosensor combines the outstanding electrochemical properties of diamond as a transducer with the controlled bonding of DNA molecules to the tips of nanowires by use of an electrochemical attachment scheme. Nucleic acid molecules are bonded in this way and dispersed to the transducer, giving rise to optimized hybridization kinetics of DNA. Negatively charged redox mediator molecules (Fe(CN)63−/4−) are applied for DNA-hybridization sensing. Voltammetric detection of DNA hybridization by differential pulse voltammetry is performed with respect to its sensitivity and reproducibility. On a sensor area of 0.03 cm2, a detection limit of 2.0 pM is achieved. As for the chemical stability of the DNA bonding to the diamond nanowires, no degradation over 30 hybridization/denaturation cycles could be detected. By use of this dilute DNA arrangement, single-base mismatch discrimination is achieved. Under the same conditions, smooth diamond modified with phenyl is not suitable for amperometric DNA sensing.