Due to its low growth rate and its fastidious nature, Mycobacterium tuberculosis is difficult to identify. Its rapid and sensitive detection is, however, critical for the control of tuberculosis. Molecular biology, and more recently electrochemical technology, have been exploited for the detection of this pathogen. In the present study, a novel DNA biosensor was developed for the highly sensitive detection of the specific DNA insertion sequence IS6110 of M. tuberculosis, using reduced graphene oxide-gold nanoparticles (rGO-AuNPs) as a sensing platform and gold nanoparticles-polyaniline (Au-PANI) as a tracer label for amplification. Reduced graphene oxide, which has a large surface area, provided a biocompatible matrix. Gold nanoparticles were electrodeposited on the surface of the rGO modified electrode, which not only increased immobilisation of the capture probe but also promoted electronic transfer. The Au-PANI nanocomposite exhibited good biocompatibility and excellent electrochemical activity. It was therefore used as a tracer label for electrochemical detection, which provided a simple preparation process for a signal-on DNA biosensor. With the excellent electroactivity of the Au-PANI nanocomposite, the resulting DNA biosensor exhibited high sensitivity for the detection of M. tuberculosis over a broad linear range, between 1.0 × 10(-15) and 1.0 × 10(-9) M. The DNA biosensor showed good stability and high specificity and provides a new strategy for clinical M. tuberculosis diagnostics and probably also for pathogenic bacteria in general.