Background: Thermal stability (TS) is a part of the BCG vaccine characterization by which the consistency of process in BCG vaccine production could be confirmed. To enhance the TS of the vaccine, some prevalent stabilizers in different concentrations were added to the final formulation of BCG bulk prior to Freeze-drying process. We found a formulation more effective than the current stabilizer for retaining the higher viability of lyophilized BCG vaccine produced by Pasteur Institute of Iran. Methods: In the design of experiments using Taguchi method, lactose, trehalose, glucose, dextran, and monosodium glutamate were added to the final formulation of BCG bulk prior to freeze-drying process. Viability of the samples was determined by counting the colony forming unit. Results: Maximum signal-to-noise ratio equal to maximum TS and viability was obtained by adding lactose, dextran, and glutamate in defined concentrations. Conclusion: Adding the stabilizers had a significant impact on TS of BCG vaccine to meet the quality requirements.