This study evaluated the immunogenicity of the Bacillus Calmette-Guérin (BCG) vaccine in a guinea pig model to refine preclinical assessment methods. 24 guinea pigs were divided into four groups for immunohistochemical, histopathological, and molecular analyses, including qRT-PCR and ELISA. The ELISA results revealed significant elevations in interleukin 2 (IL-2), interferon-gamma (IFN- ), and tuberculosis-specific antibodies in vaccinated guinea pigs, particularly γ notable after 6 weeks. Although lung cytokine levels remained unchanged, spleen gene expression showed significant differences in interleukin-17, interleukin-12, interleukin-1β, and C-X-C motif chemokine ligand 10 after 6 weeks. Immunohistochemistry revealed peak IL-2 expression at 8 weeks and significant IFN-γ and TNF-α expression at 6 weeks. This study confirmed the effectiveness of BCG vaccine in guinea pigs, providing crucial insights for future tuberculosis vaccine development and standardizing immune response indicators.