The invention discloses a preparation method of water soluble microspheres. Oil solubility nano-particles are dissolved into an organic solvent to obtain an oil phase, water-soluble hydrosulphonyl containing or disulfide bond biomacromolecules are dissolved into water to obtain a water phase; the oil phase and the water phase are premixed, and a crude emulsion is obtained; the crude emulsion is subjected to the cavatition through the physical stimulation means, biomacromolecule is subjected to a crosslinking reaction, and the biological macromolecule shells wrap the surfaces of the nano-particles. The preparation method is simple and mild in reaction conditions, the obtained microspheres have the good water solubility, the grain size is controllable, the grain size is uniform, the stability is good, the non-specific adsorption is low, the biological compatibility is good, the coupling marking is easy, and the method can be widely used for the fields of bioanalysis, environment analysis, food safety testing, sample separation and purification, medical examination, imaging, biological medicine research and the like.