The invention relates to a busulfan injection and a preparation method thereof. The invention relates to the busulfan injection which contains busulfan and a medicinal solvent, wherein the medicinal solvent is a combination of N,N-dimethylacetamide and polyethylene glycol 400 at the volume ratio of 1:(1.5-2.5); and 1ml of the busulfan injection contains 5-7mg of busulfan. The busulfan injection provided by the invention has excellent pharmaceutical properties such as excellent chemical stability. The busulfan injection provided by the invention is suitable for being combined with cyclophosphamide to serve as a preparative regimen before allogeneic hemopoietic progenitor cell transplantation of chronic myelogenous leukemia.