The invention belongs to the field of medicinal preparations for animals, and relates to an injectable Isatis Root in-situ gel preparation, and a preparation method thereof. The injectable Isatis Root in-situ gel preparation comprises, by weight, 30.0-75.0 parts of an Isatis Root extract liquid, 14.5-31.5 parts of poloxamer 407, 0.7-9.5 parts of poloxamer 188, 0.02-10.0 parts of a high-molecular retarding agent, 0.01-10.0 parts of a solubilizing stabilizer, a proper amount of a pH adjusting agent and 0.01-3.0 parts of an antiseptic, and each milliliter of the obtained preparation equals to 1.5-5.0g of a dried raw medicine. The preparation is a free-flowing liquid at room temperature, forms a semisolid gel after intramuscular or hypodermic injection, is slowly released, realizes only one-time administration for one treatment course, and has the advantages of simple preparation, convenient administration and definite therapeutic effect. The preparation has anti-virus, anti-bacterium, anti-endotoxin and immunoloregulation effects, and is suitable for controlling diseases of animals comprising pigs, cattle, sheep, dogs, cats and the like.