The invention discloses Chicken semen diluent as well as a preparation method and application thereof, and belongs to the technical field of animal breeding. Chicken semen is diluted for normal temperature preservation and artificial insemination. The Chicken semen diluent takes sterile distilled water as a solvent and contains fructose, citric acid, sodium glutamate, potassium chloride, dipotassium hydrogen phosphate, Tris, bovine serum albumin, VB12 (Vitamin B12), streptomycin and penicillin. The preparation method comprises the following steps of: dissolving the fructose, citric acid, sodium glutamate, potassium chloride, dipotassium hydrogen phosphate, Tris, bovine serum albumin and VB12 into a small amount of sterile distilled water under the sterile condition, adding the penicillin and the streptomycin, adding the sterile distilled water, mixing uniformly to obtain the Chicken semen diluent, and preserving at the temperature of 4 DEG C; and preheating the Chicken semen diluent to the temperature of 37 DEG C, and mixing the Chicken semen diluent and Chicken semen, wherein the mass ratio of the Chicken semen to the Chicken semen diluent is (1:1)-(1:2). The Chicken semen diluent has the advantages of simplicity and convenience in preparation, convenience in material acquisition, low cost and easiness in popularization.