The invention provides a breast injection medicament for resisting cow mastitis and a preparation method thereof. The breast injection medicament comprises raw materials and a cosolvent, wherein the raw materials include active ingredients and auxiliary materials; the active ingredients include the following components in parts by weight: 0.5-2.0 parts of ceftiofur hydrochloride, 1-10 parts of cloxacillin sodium and 0.05-0.25 part of prednisolone; the auxiliary materials include the following components in parts by weight: 0.5-2 parts of dispersing agent, 0.15-2 parts of antioxidant, 0.05-0.5 part of preservatives and 1-7 parts of emulsifier; and the cosolvent is cottonseed oil. The method comprises the following steps: heating 30-35% of total amount of cottonseed oil, adding the antioxidant, the preservatives, the emulsifier and the dispersing agent into the cottonseed oil, and stirring; mixing ceftiofur hydrochloride, cloxacillin sodium and prednisolone and 10-20% of total amount of cottonseed oil; mixing the mixture, adding the remaining cottonseed oil, emulsifying and cooling and subpackaging. The breast injection medicament has the effects of being fewer for in vivo residues, short in retention time, and good in healing effect on cow mastitis disease, and the like.