The invention discloses a preparation method of horseradish tree leaf wine, including the following six steps: raw material treatment, alcohol fermentation, skin and residue separating and squeezing,malolactic fermentation, natural clarificant clarifying and precipitation separating treatment and filtering and bottling. The preparation method can effectively improve the power and speed of wine fermentation, avoid the slow and stagnation of alcohol fermentation and increase the rich flavor and mouthfeel of the wine by adding horseradish tree leaf powder to the wine soaking and fermenting process to utilize amino acids, vitamins, minerals and other nutrients of the horseradish tree leaf powder as natural yeast nutrients; at the same time, active ingredients in the horseradish tree leaf powder are extracted during the fermentation process, including polyphenols, flavonoids, astragalin, chlorogenic acid, carbamate, carbamate sulfide, etc., increasing the activity of the wine in lowering blood glucose and blood lipid, resisting bacteria, diminishing inflammation, resisting cancer, resisting oxidation, resisting ulcer, etc.