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A novel strategy for preparation of aptamer-based organic-silica hybrid affinity monolithic column via “thiol-ene” click chemistry coupled with simple spectrophotometry using a chromogenic substrate for human α-thrombin enriched by the aptamer affinity column were developed successfully.Due to the large specific surface area of the hybrid matrix and the simpleness,rapidness and high efficiency of click reaction,the average coverage density of aptamer on the organic-silica hybrid monolith reached 420 pmol/μL.Thrombin can be retained with high specificity by this aptamer affinity column and detected sensitively with a detection of limit of 0.01 μM by chromogenic assay.Furthermore,the extraction recovery of thrombin in human serum was 91.8% with a relative standard deviation of 4.0%.These results indicated that the “thiol-ene” click chemistry provided a novel technique to immobilize aptamer on organic-inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to achieve highly selective recognition of trace proteins.