Silica microspheres are easily agglomerated in anhydrous and aqueous media, which would seriously affect the properties and correlative studies of these particles. In order to effectively investigate and improve the characteristics of silica microspheres, the de-aggregation technology is indispensable. Through analysis of the mechanism of this aggregation phenomenon, a method was proposed to de-agglomerate the large-scale-agglomerated silica particles by ten steps. Experimental results observed by Nikon microscopy indicate that this method could greatly improve the dispersity and stability of the particles.