The invention provides a preparation method of a Dicranostigma leptopodum extract perfusate. The method comprises the following steps: adding water to a Dicranostigma leptopodum extract, decocting, mixing filtrates, and concentrating to obtain a concentrate; and extracting with ethanol, removing ethanol, adding benzyl alcohol and Tween-80 to the above obtained ethanol removed medicinal liquid, adding injection water to a full dose, and sterilizing to obtain the Dicranostigma leptopodum extract perfusate. The invention also provides an application of the perfusate in the preparation of medicines for treating cow mastitis. The Dicranostigma leptopodum extract perfusate adopting the Dicranostigma leptopodum extract as an effective component has very good heat clearing, detoxifying, bacterium resisting, inflammation eliminating and virus resisting effects. The perfusate is a pure traditional Chinese medicinal preparation, contains no chemical components, and is in favor of the safety of dairy products. Products produced through extraction and preparation integrated production have the advantages of stable properties and controllable quality, and are in favor of industrial production. The perfusate has the advantages of definite effect, no residual or toxic side effects, and realization of use in the lactation period as an effective medicine for treating clinic mastitis of cows, and becomes a good substitute of antibiotics.