Objective To assess the potential of whole blood IFN-γ assay for diagnosing mycobacterium in rhesus macaques. Methods Firstly, basic serum IFN-γ concentrations of TST-negative and -positive rhesus macaques were detected. Then, heparinized whole blood from TST-negative and -positive rhesus macaques was incubated with PBS and 200 IU bovine-PPD (tuberculin purified protein derivative) for about 24 h, respectively. The supernatant plasma were harvested and used to determine the concentrations of IFN-γ. The results of plasma IFN-γ concentrations and stimulation index (SI) were used to analyze the diagnostic potential of the whole blood IFN-γ assay. Results The basic serum concentrations of IFN-γ for the TST-positive monkeys were significantly higher than that of the TST-negative macaques, showing a high coefficient of variation. There was no significant effect on the production of IFN-γ in the TST-negative macaques. While significantly elevation of IFN-γ concentrations was found in stimulated plasma of TST-positive macaques (P<0.01). The SI of TST-positive macaques was significantly higher than the TST-negative ones. ROC curve analysis revealed that IFN-γ concentrations and SI could be used as evaluation index of whole blood IFN-γ assay. Conclusions Based on a small sample experiment we have demonstrated that whole blood IFN-γ assay may be one possible auxiliary diagnostic method for tuberculin skin test.