Abstract: Provided are a traditional Chinese medicine ointment for preventing and treating mastitis in dairy cows and a preparation method thereof, belonging to the technical field of veterinary traditional Chinese medicines. The traditional Chinese medicine ointment includes raw materials in parts by mass: 20-30 parts of dandelion, 20-30 parts of herba patriniae, 10-16 parts of Caulis Lonicerae Japonicae, 0.5-1 part of borneol, 0.03-0.05 part of polypeptide, 40-50 parts of auxiliary emulsifier, 40-50 parts of liquid paraffin and 100-130 parts of white vaseline. An amino acid sequence of the polypeptide is: Phe Phe Arg Lys Val Leu Lys Leu Ile Arg Lys Ile Trp Arg (SEQ ID NO. 1). In the application, dandelion, herba patriniae, Caulis Lonicerae Japonicae and borneol 1 are mixed, and polypeptide is added at the same time to prepare ointment.