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Molecularly imprinted polymers (MIPs) have been utilized as biorecognition elements in various fields such as separation, sensing and imaging due to their easy accessibility and high binding capacity to target molecular analytes. To expand the application scope of MIPs and widen their range of targets to microorganisms, we have developed a methodology for the synthesis of MIP shells on the surface of polystyrene microparticles (MPs) with a binding affinity towards E. coli OP50 as a bacterial surrogate. Monodisperse MIP-based core–shell microparticles (MIP-MPs) with controllable shell thickness ranging from 0.25 μm to nearly 3.5 μm were produced using a stepwise polymerization method. Optical and fluorescence microscopy as well as scanning electron microscopy were used for characterization of MIP-MPs and MPs coated with non-imprinted polymers (i.e., NIP-MPs) under various timing and temperature settings. E. coli OP50 imprinting and rebinding experiments were conducted using the MIP-MPs with a suitable MIP shell thickness of 2–3 μm. Capturing experiments were carried out with different concentrations of NIP- and MIP-MPs (i.e., 102, 103 and 104 particles/mL) to investigate the uptake ratio of bacteria (104 cells/mL) and its particle dose-dependency. The optimum uptake ratio of E. coli was approximately 74%, which was achieved using 103 MIP-MPs/mL. NIP-MPs had reduced bacteria recovery and results were statistically lower than MIP-MPs, showing the affinity of our MIP shells towards microorganisms. The results reported here provide a new fabrication methodology and binding efficiency of core–shell MIP-MPs to bacteria, and can create the basis for the fabrication of various imprinted coating layers on spherical substrates with potential applications in bioseparation and point-of-care sensing. [Display omitted] • Polystyrene microparticles (MPs) with molecularly imprinted polymer (MIP) shells. • MIPs composed of four monomers for enhanced imprinting of bacteria cells on MPs. • Two-step temperature-dependent polymerization to control MIP shell thicknesses. • Optimum MIP-MPs to bacteria ratio of 1–10, showing high-level MIP imprinting. • MIP-MPs reached 74% bacteria capturing efficacy improving the state of the art. [ABSTRACT FROM AUTHOR]