The invention belongs to the field of special pharmaceutical preparations for animals, and relates to an in-situ gel preparation for enrofloxacin injection and a preparation method thereof. The preparation comprises the following components in parts by weight: 5.0-20.0 parts of enrofloxacin, 14.5-32.5 parts of P407, 0.5-10.5 parts of P188, 25.0-90.0 parts of water, 0.01-10.0 parts of a high molecular retardant, 0.01-3.0 parts of a preservative and appropriate amount of a pH adjustor. The preparation provided by the invention is a freely flowing liquid at room temperature, and forms a semi-solid gel through dosage by intramuscular injection or subcutaneous injection, so that the preparation is just delivered once in a course of treatment. The preparation has the advantages of stable property, long lasting time for delivery, exact curative effect, strong animal adaptability, no toxic and side effects and adverse reaction and the like, and is simple to prepare and convenient to delivery. The preparation provided by the invention has broad-spectrum antibacterial effect, and can be used as a medicine for preventing and treating diseases of animals such as pigs, sheep, dogs, cats and cattle.