ZnSe quantum dots (QDs) have attracted attentions due to small size and strong fluorescence. However, ZnSe QDs are easy to agglomerate and insoluble in aqueous solutions, which limits their practical applications. Herein, we report a novel strategy for synthesizing highly dispersed and water-soluble ZnSe QDs by electron beam radiation with the aid of EDTA within only a few minutes at room temperature without any reduction reagents. In the reaction system, EDTA plays an important role in controlling the quantum size of crystal growth, inhibiting the agglomeration of ZnSe QDs and improving the water solubility. ZnSe QDs exhibit a large bandgap of 4.3 eV, significantly blue shifted compared with bulk ZnSe due to the quantum confinement effect. Constructed the structural model by MS, the nucleation reaction and growth mechanism of ZnSe QDs are proposed. Electron-beam irradiation can be widely used to synthesize water-soluble semiconductor QDs with promising application in biological imaging.