The invention discloses a preparation method of moxifloxacin impurity F. The method comprises the following steps of: alkalifying moxifloxacin hydrochloride with sodium bicarbonate solution to get moxifloxacin free alkali, treating the moxifloxacin free alkali with methylation and alkaline hydrolysis reaction, and finally, obtaining high-purity moxifloxacin impurity F by recrystallization. The preparation method has the characteristics that the synthetic routes are simple and short, operations are simple and convenient, the obtained impurity product has higher purity, and the preparation method can be used for moxifloxacin impurity reference substance research, and so on.