Submicron-scaled molecularly imprinted polymer (MIP) particles for 17--estradiol were prepared by precipitation polymerization method using 2-(trifluoromethyl) acrylic acid as functional monomer and trimethylolpropane trimethacrylate as cross-linker in acetonitrile. The obtained submicron-scaled MIP particles were subsequently packed into stainless steel columns and the chromatographic characteristics of the MIP stationary phase were investigated. Interestingly, it was found that tandem MIP columns of 17--estradiol-based MIP with two different ratios of the template could achieve almost complete separation of isomers (17--estradiol and 17--estradiol) with a good separation factor (2.45). Morphological characteristics and adsorption kinetics of the submicron-scaled MIP particles, effect of flow-rate, injection amount, and column temperature on separation were also investigated in detail. Results indicated that the submicron-scaled MIP particles had both good selectivity and high affinity to the template molecule 17--estradiol