The invention discloses a preparation method of a coating type chloracetylated SiO2 fluorescent nanosphere. A double-bond allyl fluorescent probe, methacrylic acid and a silane coupling agent KH151 are used as raw materials; polyreaction is performed to obtain a fluorescent polymer; through the effect of chemical bonds, the fluorescent polymer is grafted onto the surface of an SiO2 nanosphere so as to obtain the coating type chloracetylated SiO2 fluorescent nanosphere through preparation. The prepared coating type chloracetylated SiO2 fluorescent nanosphere has the advantages that the morphostructure is stable; the monodispersity is good; the luminous efficiency is high; the biocompatibility is high; the carboxylation functional groups are realized; the firm -Si-O-Si- bond also exists; theproduct fluorescence leakage and quenching are avoided.