The invention relates to a preparation method of a zinc oxide microsphere with a nanoflower-shaped microstructure. The method comprises the following steps of: (1) preparing a solution, to be specific, preparing a soluble zinc salt into an aqueous solution, and preparing a soluble alkali into an aqueous solution; (2) mixing the solutions, to be specific, mixing the soluble zinc salt aqueous solution with the soluble alkali aqueous solution at the room temperature in the volume ratio of 1:10-10:1, slowly pouring an aqueous solution of a soluble zinc salt into the aqueous solution of the soluble alkali, and stirring to obtain a clarifying solution; and (3) preparing a product, to be specific, pouring a weakly-acid solution into the clarifying solution obtained in the step (2), wherein the volume ratio of the weakly-acid solution to the clarifying solution is 1:10-10:1, stirring to obtain a turbid solution containing a large quantity of products, repeatedly washing with deionized water and absolute ethyl alcohol, and drying in a baking oven to obtain a final product. Compared with the prior art, the method has the advantages of capability of rapidly, easily and conveniently preparing a product under the room temperature condition, high yield, low cost, environmental friendliness, wide raw material selection range and contribution to industrial production.