The invention discloses a preparation method of an agarosebonded silica gelhybridized monolithic column. The preparation method comprises the steps that tetramethyl orthosilicate, agarose, polyethylene glycol and urea are mixed under an ice-bath condition to prepare a prepolymerization system by adopting a sol-gel method, then the prepolymerization system is put in a quartz capillary tube to prepare an affinity monolithic column, and finally the affinity monolithic column is subjected to three-step modification to obtain the agarosebonded silica gelhybridized monolithic column containing amide groups on the surface. The preparation method of the agarosebonded silica gelhybridized monolithic column has the advantages of being simple in operation and making the surface of the prepared monolithic column convenient to modify, the problem that an immunoaffinity column using agarose gel as a carrier is not easily stored and easily produces swelling is solved, and the prepared monolithic column has the advantages of being good in mechanical strength, stable in framework structure, good in permeability, uniform in texture, good in hydrophily and reproducibility and the like and has high selectivity and high concentration rate to biomolecule.