The invention discloses a mercaptobenzimidazole sulphonic acid type organic-inorganic hybridization integral column, and a preparation method and application thereof. sodium 2-mercaptobenzimidazole-5-sulfonate is used as a functional monomer to be subjected to ultrasonic uniform mixing with inorganic monomers, hybridization monomers, initiators and pore-foaming agents in an ice bath; the materials are injected into vinyl modified quartz capillary; through sulfhydryl-alkene clicking and one-pot reaction, hot triggering polymerization, cleaning and drying, the mercaptobenzimidazole sulphonic acid type organic-inorganic hybridization integral column is obtained. The sulfhydryl-alkene clicking chemical reaction is used for bonding mercaptobenzimidazole sulphonic acid type compounds into a silicon gel framework; the prepared mercaptobenzimidazole sulphonic acid type organic-inorganic hybridization integral column framework has good permeability, porosity and chemical and mechanical stability; the separation of biological molecules of organic small molecules, nucleoside, alkaloid, anesthetics or protein and the like can be realized.