Fluorescent microspheres are prepared by attaching self-assembled polydiacetylene (PDA) vesicles withcarboxyl side groups onto the substrate amino-modified poly(glycidylmethacrylate) (APGMA)microspheres. The characterization by SEM, confocal microscopy and flow cytometry demonstrated thatthe final resulting microspheres are highly uniform both in size (with a diameter of 5 mm) and influorescence emission (coefficient of variance < 3%). The Brunauer–Emmett–Teller (BET) surface area ofthese spheres is 114 m2.g-1. In addition, there are evenly distributed pores with an average size of 20.6nm on the spheres. These spheres are found to have good thermal stability and photostability, and donot suffer from fluorophore leaching. Fluorescein isothiocyanate (FITC) labelled bovine serum albumin(BSA) as a representative biomolecule can be easily attached onto the fluorescent microspheres. Allthese characteristics possessed by the APGMA–PDA spheres allow them to be directly used as carriers ofbiomolecules in lab-on-a-chip immunoassay systems.