The present invention provides a preparing method of ZnO quantum dot. Firstly, processing surface treatment to a substrate, then forming a layer nano-sphere assembled by itself by czochralski method in deionized water, the nano-sphere is used as a template for preparing ZnO quantum dot; further, depositing a layer ZnO film which thickness is 5nm-50nm by electron beam evaporation under the conditions of temperature between 25-100 DEG C, 10<-4>-10<-6> Torr vacuum; finally, dipping in toluol, and vibrating 2-5 min by ultrasonic to remove the nano-sphere in the template. The ZnO quantum dot prepared by the method has large growth area, regular arrangement, even distribution. The preparing method simply, economically and effectively produces ZnO quantum dot having larger range, and order. The ZnO oxide semiconductor energy gap is wide, direct wide band gap is transmitted, and exciton binding energy is large, the ZnO quantum dot material becomes expectations of high quantum dot laser and nan0-laser by adding the quantum confinement effect of the quantum dot.