Objective: Activating and amplifying γδΤ cells with Mycobacterium tuberculosis low molecular peptide antigen (Mtb-Ag), in order to investigate the expression of CD69 molecules on γδΤ cellular surface. Methods: Activate health human peripheral blood mononuclear cells (PBMC) separately obtained, PBMCs were stimulated with Mtb-Ag and further isolate positive cells by immuno-magnetic beads selection, measure the proportion of γδΤ cells in the PBMCs by fluorescent monoclonal TCR γδΤ-PE staining and flow cytometry. Then, measure the expression of CD69 molecules in first stimulation and re-stimulation of γδΤ cells by γδ-PE/CD69FITC double staining. Results: The proportion of γδΤ cells were 4.9% in freshly isolated from PBMC, 69.2% after 10 days of Mtb-Ag activation, and 99.3% after immuno-magnetic beads selection. After 24 hours, the expression of CD69 molecules in γδΤ cells with initial Mtb-Ag stimulation arrived at peak at 75.2%. 6 hours later, in the second stimulation, it peaked at 72.0%. Conclusion: Mtb-Ag can specifically stimulate the proliferation of γδΤ cells in the PBMC. Both its initial and the second stimulation can specifically activate γδΤ cells.