The invention provides a tacrolimus ophthalmic preparation and a preparation method thereof, and belongs to the technical field of pharmaceutical preparations. According to the present invention, thetacrolimus ophthalmic preparation has a micro-emulsion-like structure, is clarified and transparent, has good patient compliance, is prepared from tacrolimus, oil for injection, a surfactant, a stabilizer, an osmotic pressure regulator, a preservative, a pH value regulator, a tackifier and water for injection, and is used for preventing and treating immunological rejection after corneal transplantation or immunological corneal xerosis and conjunctival xerosis. According to the present invention, with the tacrolimus ophthalmic preparation, the problem of the water insolubilization of tacrolimusis solved, the tacrolimus is firstly prepared into the clarified and transparent eye drops with the micro-emulsion-like structure, the irritation on the eyes by the existing tacrolimus-suspended ophthalmic eye drops can be substantially reduced, and the patient compliance can be improved, such that the tacrolimus ophthalmic preparation of the present invention is suitable for the long-term use byimmunological rejection after corneal transplantation, corneal xerosis and conjunctival xerosis, and other patients with indications.