The present invention provides a preparation method of an uniform-size nanoparticle fluorescent microsphere. The density of fluorescent nanoparticle colloidal aqueous solution of the microsphere is adjusted to the micro-mol level so as to obtain the colloidal solution evenly scattered in the aqueous solution; the evenly scattered colloidal solution in the aqueous solution is injected into oil-phase solvent through capillary at the speed of 0.5 to 1.25 percent Vs/min so as to form droplets and Vs is the volume of the oil-phase solvent; the oil-phase solvent adopts bi- or multi-mixture with thesolubility between 2 to 10 percent and the density equal to the density of water; containers and reflux pipes adopt the hydrophobic material; the water in droplets is absorbed by the oil phase and the nanoparticles form a dense sphere; an absorbent continuously absorbs the water dissolved in the oil phase, so that the water content in the oil phase is far lower than the saturation; the nanoparticle polymer with the size from dozens of nano to hundreds of nano, the spherical height higher than 5 percent and the size error less than 10 percent is obtained through regulating the density of the quantum pot colloidal solution and the velocity of the reflux pipe.