Fluorescent molecularly imprinted polymers (MIPs) have received considerable attention for their promising applications in sensing, imaging and diagnostics. Different synthetic methods have been reported to prepare fluorescent MIPs. Here, one-pot polymerization method was developed to synthesize fluorescent MIP nanoparticles (NPs) bearing a suitable fluorescent label as the fluorescence acceptor. The MIP NPs can emit strong fluorescence based on fluorescence resonance energy transfer (FRET) when they bind to analytes acting as the fluorescence donor. The one-pot synthetic method is straightforward, less time-consuming and easy to optimize the loading of fluorescence acceptor. Additionally, a preliminary microfluidic reactor was designed for preparing such fluorescent MIP NPs with more uniform size in a continuous process for the first time. The fluorescent MIP NPs can be used for selective detection of the analytical target in a FRET-based turn-on fluorescence assay which is easy to carry out with no tedious separation steps. (c) 2018 Elsevier Ltd. All rights reserved.