We demonstrate the facile preparation of highly luminescent Cd_1−xZn_xSe_1−yS_y quantum dot (QD)-encoded poly(styrene-co-ethylene glycol dimethacrylate-co-methacrylic acid) beads (PSEMBs) in a straightforward and reproducible manner. The monodisperse mesoporous PSEMBs are first swelled in chloroform. Afterwards, the reaction precursors, composed of Cd, Zn, Se and S, are impregnated into the microspheres. Subsequently, the Cd_1−xZn_xSe_1−yS_y QDs are synthesized directly within the polymer beads by thermal decomposition. The results show that a wide range of emission wavelengths (490 nm–606 nm) with a narrow full width at half maximum (FWHM) (<40 nm) is obtained by changing the ratios of the precursors. Confocal microscopy images illustrate that highly uniform, bright fluorescent beads are obtained and the QDs have infiltrated into the entire microsphere. The resulting QD barcodes exhibit remarkable stability against solvent induced QD leaching and chemical induced fluorescence quenching. The QD-encoded PSEMBs are found to be photostable in phosphate buffered saline (PBS) (pH 7.4) for at least 3 weeks. Immunoassay performances for human IgG detection indicate that the QD barcodes can be successfully applied to high-throughput multiplexed biomolecular assays.