The invention relates to a preparation method of a micro-nano fluorescent dye microsphere in the field of micro-nano material preparation. The method comprises the following steps: dissolving a dye ina solvent to prepare a precursor solution, filling the precursor solution into an injector, and under the action of the pushing force of the injector, allowing the precursor solution to form suspended conical liquid drops, namely Taylor cones at the tail end of a spray head. The suspended conical liquid drops are crushed into micro droplets under the action of electric field force, the micro liquid drops are stretched at a high speed through the electric field force in a short distance, the solvent is volatilized, and finally the micro liquid drops are deposited on a receiving electrode plateand dried by a drying box to obtain the micro-nano fluorescent dye microsphere. The method has the advantages that the cost is low, the process is simple, production parameters can be changed for size adjustment according to needs, and the method is suitable for continuous production.