The invention relates to a nanometer imprinted microsphere for conducting identification and fluorescent quantitation on a pesticide. The nanometer imprinted microsphere for conducting identificationand fluorescent quantitation on the pesticide is of a multi-layer structure, and is characterized by being composed of a SiO2 nanometer microsphere core and three layers of shells, wherein the SiO2 core is covered by a rare earth ion layer, the rare earth ion layer is covered by a silicon dioxide layer, and a molecularly imprinted polymer layer is formed on the silicon dioxide layer; the molecularly imprinted polymer layer is a molecularly imprinted polymer layer of the pesticide. The invention further relates to a preparation method of the nanometer imprinted microsphere.