This study reports the development of a new PCR-free device, using IS6110 gene as biomarker, for Tuberculosis (TB) diagnosis. An arginine film (ARGFILM) was used to prepare the biosensor platform. MT-probe was immobilized on this biosensor platform to identify IS6110 gene. This gene is an excellent biomarker for Mycobacterium tuberculosis (MT). Electrochemical analyses were carried out using differential pulse voltammetry method (DPV) by methylene blue (MB) reduction signal measurement before and after hybridization either between probe and synthetic target or extracted DNA from clinical sputum samples. The optimization study of MT-probe immobilization on modified-electrode surface showed that the best probe concentration was 15 μM. The analytical analysis of hybridization assays was performed using different concentrations of synthetic MT-target (15–500 nM). The linear response was between 15 and 100 nM and the detection limit was 4.4 nM. The biosensor performance was also investigated with extracted DNA from sputum samples (PCR-free). The results showed that the biosensor was able to detect the MT from samples, exhibiting a high sensitivity and satisfactory selectivity. Thus, these results allow for the possibility of developing a portable detection device for effective diagnosis of TB patients.