Monolithic molecularly imprinted column was prepared by an in-situ therm-initiated copolymerization and the effects of essential preparation conditions such as polymerization mixture composition and polymerization condition was investigated. The results showed that the selection of correct porogenic solvents and appropriate polymerization conditions are crucial for the preparation of the monolithic stationary phases. The separation efficiency was only extremely weakly dependent on flow rate and hydrogen-bonding interaction played an important role in the retention and separation. Compared with conventional particle columns and bulk molecular imprinted polymer column, the monolithic column exhibited good stability, ease of regeneration, high separation efficiency, and fast analysis.