The invention discloses a finless eel sperm diluent and a preparation method thereof. The finless eel sperm diluent is prepared from sodium chloride, potassium chloride, sodium bicarbonate, calcium chloride, and sodium hydrogen phosphate according to a ratio. The preparation method comprises the steps of A, preparing sperm diluent mother liquor: preparing sperm diluent mother liquors respectively containing 1mmol of sodium chloride, 1mmol of potassium chloride, 1mmol of sodium bicarbonate, 0.5mmol of calcium chloride, and 0.5mmol of sodium hydrogen phosphate in per ml, and storing the mother liquors in a refrigerator, wherein the mother liquor containing sodium hydrogen phosphate is prepared by the steps of calculating the weight of sodium hydrogen phosphate for preparing the mother liquor according to the concentration and volume of the mother liquor and the molecular weight of sodium hydrogen phosphate, weighing sodium hydrogen phosphate, and putting sodium hydrogen phosphate into a beaker; and B, preparing the sperm diluent: measuring and preparing the diluent from the mother liquor containing each component, evenly mixing each component, evenly mixing the prepared diluent in a flask, diluting to a certain volume by distilled water, and storing the prepared diluent in the refrigerator for use, wherein the amount of each mother liquor is calculated according to the concentration of each component in the formulation of the diluent and the volume of the diluent. The finless eel sperm diluent is reasonable in formulation, convenient to use, and suitable for diluting the finless eel sperm; the sperm is effectively diluted; the artificial insemination cost is reduced, and the fertility rate is improved.