Abstract: The aim of the present study was to apply a small dimension, low cost and simple electrochemical biosensor for the detection of nucleic acid related to tuberculosis (TB) using Methylene Blue (MB) as the electroactive intercalator. The MYC Probe was immobilized on the Screen-Printed Carbon Electrode (SPCE) surface via non covalent attachment. The electrochemical biosensor of the DNA hybridization was evaluated using Differential Pulse Voltammetry (DPV) for measuring the interaction of MB with DNA oligonucleotides on SPCE using a PalmSens Electrochemical Portable Apparatus. Parameters affecting the response of the voltammetric signals towards detecting nucleic acid were optimized including concentration of MYC Probe, immobilization time of MYC Probe, hybridization time of immobilized MYC Probe, concentration of MB and time of MB accumulation. Various synthetic oligonucleotides of Mycobacterium tuberculosis (M. tuberculosis) were used such as MYC Probe, MYC Target, MYC Non-complementary and MYC Mutation for the DNA hybridization detection on SPCE. Differences in the voltammetric signals of MB were observed between MYC Probe and Polymerase Chain Reaction (PCR) amplified products for the detection of M. tuberculosis DNA. It is concluded that MB could be used as an effective electroactive hybridization indicator in the development of DNA biosensors for detection of M. tuberculosis DNA.