The invention belongs to the field of pharmaceutical formulations specially used for animals, and relates to an in-situ gel formulation for florfenicol injection and a preparation method thereof. The in-situ gel formulation is prepared from antibacterial drugs, an in-situ gel substratum and other indispensable auxiliary materials, wherein the antibacterial drugs are specially used for animals; the in-situ gel substratum is accredited in the field. The preparation method is illustrated.The in-situ gel formulation disclosed by the invention is freely flowing liquid at room temperature, forms the semisolid gel after intramuscular injection or hypodermic injection administration, realizes only one-time administration within one treatment course, has the advantages of easiness for preparation, stability in property, convenience for administration, long administration effect lasting time, definite treatment effect, high animal adaptability, no toxic or side effect or adverse reaction, and the like, achieves the broad-spectrum antibacterial effect and is suitable for preventing and treating various sensitive bacteria infection of the animals, such as pigs, sheep, dogs, cats, cattle, and the like.