Objective To investigate the synergistic and bactericidal effects of ultrasound combined with slow-release PLGA nanoparticles (LEV-NPS) on Bacille Calmette-Guerin (BCG) of attenuated bovine tuberculosis in vitro and in vivo. Methods Levofloxacin PLGA sustained release nanoparticles were prepared by double emulsification method. Bacterial activity and morphological and structural changes were evaluated by plate colony counting and scanning electron microscopy (SEM). Subcutaneous tuberculosis granuloma infection model was established, and the therapeutic effect was evaluated by continuous monitoring of granuloma volume change. Results LEV-NPs was spherical, uniform in size and had obvious sustained release properties. Compared with the control group and LEV alone group, ultrasound combined with LEV-NPS significantly reduced bacterial activity, destroyed bacterial structure, and reduced subcutaneous tuberculous granuloma volume, with statistically significant differences (P<0.05). Conclusions Ultrasound mediated slow-release PLGA nanoparticles (LEV-NPS) can significantly improve the bactericidal effect of LEV against BCG of attenuated bovine tuberculosis, which have significant synergistic effect.