Disclosed are levoisovalerylspiramycin I, II or III, preparations, preparing methods and uses thereof. The preparations comprise levoisovalerylspiramycin I, II or III and pharmaceutically acceptable carrier and/or adjuvant, wherein the purity of levoisovalerylspiramycin I, II or III is above 90 wt%. The levoisovalerylspiramycin I, II or III has a good antibacterial activity, and the preparations include solution for injection, powder for injection or lyophilized powder for injection.