Interferon-gamma (IFN-γ) released by T cells performs very important roles in latent diagnosis of Mycobacterium tuberculosis (MTB) infections and control of tuberculosis. In this work, a multilayer screen printing indium tin oxide (ITO) detector modified with a self-assembled graphene/ chitosan (Gr-Chit) composite film followed by glutathionemodified gold nanoparticles (GSH-AuNPs), antibodies, and antigens was fabricated for highly sensitive and rapid detection of IFN-γ. The formation of the antibody-antigen complex at the electrode surface causes the impedance to increase. Under optimum conditions, the relative increased differential pulse voltammetry (DPV) peak current values are proportional to the logarithmic value of IFN-γ concentrations in a wide range of 5 to 4000 pg/mL with a low detection limit of 0.5 pg/ mL (S/N=3). The resulting IFN-γ immunosensor shows excellent detection reproducibility, good specificity, and stability. In addition, for the detection of clinical serum samples, it is well consistent with the standard enzyme-linked immunosorbent assay (ELISA) tests. Compared to the expensive glassy carbon electrode, the ITO detector can act as a disposable electrode in clinical detection due to its low cost. This study provides a lower cost, simple, and highly sensitive approach for potential application in early diagnosis of MTB infections and control of tuberculosis.