We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridization between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analytical parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples.