Molecular imprinting of cis-diol functionalized agents via boronate affinity interaction has been usually performed using nanoparticles as a support which cannot be utilized as a stationary phase in continuous microcolumn applications. In this study, monodisperse-porous, spherical silica particles in the micron-size range, with bimodal pore diameter distribution were selected as a new support for the synthesis of a molecularly imprinted boronate affinity sorbent, using a cis-diol functionalized agent as the template. A specific surface area of 158 m2/g was achieved with the imprinted sorbent by using monodisperse-porous silica microspheres containing both mesoporous and macroporous compartments as the support. High porosity originating from the macroporous compartment and sufficiently high particle size provided good column permeability to the imprinted sorbent in microcolumn applications. The mesoporous compartment provided a large surface area for the parking of imprinted molecules while the macroporous compartment facilitated the intraparticular diffusion of imprinted target within the microsphere interior. Amicrofluidic boronate affinity systemwas first constructed by using molecularly imprinted polymeric shell coated monodisperse-porous silica microspheres as a stationary phase. The synthetic route for the imprinting process, the reversible adsorption/desorption behavior of selected target and the selectivity of imprinted sorbent in both batch and microfluidic boronate affinity chromatography systems are reported.