The invention relates to the technical field of medicinal chemistry, and in particular, relates to a vincristine compound impurity, and a preparation method and an application thereof, wherein the preparation method comprises the following steps: adding acetone, acetic acid and acetic anhydride into vinca total alkali sulfate in proportion, and then stirring; slowly dropwise adding a prepared mixed solution of concentrated sulfuric acid, potassium dichromate and water, after the reaction is completed, adjusting to alkalinity, extracting, evaporating, concentrating, and purifying to obtain thevinblastine compound impurity, so as to provide reference or contrast for quantitative and qualitative analysis of vinblastine sulfate impurity detection.