Tuberculosis (TB) remains one of the leading causes of morbidity and mortality all over the world and multidrug resistance TB (MDR-TB) pose a serious threat to the TB control and represent an increasing public health problem. In this work, we report a homogeneous signal-on electrochemiluminescence (ECL) DNA sensor for the sensitive and specific detection of rpoB genes of MDR-TB by using ruthenium(II) complex functionalized graphene oxide (Ru–GO) as suspension sensing interface and ferrocene-labeled ssDNA (Fc–ssDNA) as ECL intensity controller. The ECL of Ru–GO could be effectively quenched by Fc–ssDNA absorbed on the Ru–GO nanosheets. The Ru–GO has good discrimination ability over ssDNA and dsDNA. Mutant ssDNA target responsible for the drug resistant tuberculosis can hybridize with Fc–ssDNA and release Fc–ssDNA from Ru–GO surface, leading to the recovery of ECL. Mutant ssDNA target can be detected in a range from 0.1 to 100 nM with a detection limit of 0.04 nM. The proposed protocol is sensitive, specific, simple, time-saving and polymerase chain reaction free without complicated immobilization, separation and washing steps, which creates a simple but valuable tool for facilitating fast and accurate detection of disease related specific sequences or gene mutations.