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 out. Then, heparinized whole blood from TST-negative and positive rhesus macaques was incubated with PBS and 200 IU bovine-PPD for about 24 h respectively. The supernatant plasma were harvested and used to determine the concentrations of IFN-γ. Results of plasma IFN-γ concentrations and stimulation index (SI) were used to analyses the diagnostic potential of whole blood IFN-γ assay. Results The basic serum concentrations of IFN-γ for the TST-positive were significantly higher than the TST-negative, showing high coefficient of variation. There was no significant effect on the production of IFN-γ in TST-negative macaques. While significantly elevation of IFN-γ concentrations were found in stimulated plasma of TST-positive macaques (P＜0.01). The SI of TST-positive macaques was significant higher than the TST-negative. ROC curve analysis revealed that IFN-γ concentrations and SI could be used as evaluation index of whole blood IFN-γ assay. Conclusion Conclusion based on small samples could be drawn that whole blood IFN-γ assay might be one possible auxiliary diagnostic method for TST.