Optical barcoding technology based on quantum dot (QD)-encoded microparticles has attracted increasing attention in high-throughput multiplexed biological assays, which is realized by embedding different-sized QDs into polymeric matrixes at precisely controlled ratios. Considering the advantage of droplet-based microfluidics, producing monodisperse particles with precise control over the size, shape and composition, we present a proof-of-concept approach for on-demand preparation of QD-encoded microparticles based on this versatile new strategy. Combining a flow-focusing microchannel with a double T-junction in a microfluidic chip, biocompatible QD-doped microparticles were constructed by shearing sodium alginate solution into microdroplets and on-chip gelating these droplets into a hydrogel matrix to encapsulate CdSe/ZnS QDs. Size-controllable QD-doped hydrogel microparticles were produced under the optimum flow conditions, and their fluorescent properties were investigated. A novel multiplex optical encoding strategy was realized by loading different sized QDs into a single droplet (and thus a hydrogel microparticle) with different concentrations, which was triggered by tuning the flow rates of the sodium alginate solutions entrapped with different-colored QDs. A series of QD-encoded microparticles were controllably, and continuously, produced in a single step with the present approach. Their application in a model immunoassay demonstrated the potential practicability of QD-encoded hydrogel microparticles in multiplexed biomolecular detection. This simple and robust strategy should be further improved and practically used in making barcode microparticles with various polymer matrixes. [ABSTRACT FROM AUTHOR]