The invention provides a preparation method of netilmicin sulfate freeze-dried powder for injection. The quality of the obtained netilmicin sulfate freeze-dried powder is greatly improved through the adjustment of process steps such as the adjustment of medicine adding sequences as well as the precise control over temperatures, time, pressures and other parameters, the multilayer control of filtration and the like. Thus, the storage life of the netilmicin sulfate freeze-dried powder is prolonged; the process is more normalized. As a result, the preparation method is suitable for being popularized.