The invention belongs to the technical field of animal breeding, and particularly relates to animal fresh semen low-temperature diluent as well as a preparation method and application thereof. The animal fresh semen low-temperature diluent is prepared from the following ingredients: glucose, sodium citrate, penicillin, streptomycin, enrofloxacin, yolk, defatted milk, vitamin A, vitamin D, vitaminE and water. The diluent has the advantages that Australian white sheep semen is stored for 1 to 2 days in a liquid state form under the condition of low temperature being 0 to 5 DEG C; the artificialinsemination requirement can be reached; the fresh semen storage quality is improved, so that the utilization rate of excellent rams is improved; high nanny goat conception rate is obtained; the problem of long-distance transportation after excellent ram semen collection is solved; the manufacturing method of the diluent is simple; the raw material cost is low; the operability is high; the large-scale popularization and application in the production is convenient.