Fluorescent microspheres with reactive sites for interacting with biomolecules are greatly demanded in flow cytometry based suspension array. Aiming to develop a new method for preparing fluorescent microspheres, two poly(p-phenyleneethylene) (PPE) conjugated polymers (CPs) with pedant carboxylic groups were synthesized via Sonogashira coupling and followed with hydrolysis of ester groups; then the conjugated polymers were immobilized onto monodispersed amino-modified porous poly(glycidylmethacrylate) (APGMA) microspheres via coupling reaction between carboxylic and amino groups to give APGMA-CP fluorescent microspheres. The fluorescent microspheres were found to have good photo- and thermal stability as well as negligible influence from rigorous washing. The emission was uniform all across the inner and surface of the spheres. To evaluate the effectiveness of bioconjugation on the fluorescent microspheres, fluorescein isothiocyanate isomer I (FITC) labeled bovine serum albumin (BSA) (BSA-FITC) was chosen as the representative biomolecule to react with the fluorescent microspheres to give APGMA-CP-BSA-FITC. In the flow cytometry study, fluorescence compensation between the V500 and FITC detectors (receiving signals from fluorophores excited by 405 nm and 488 nm, respectively), to remove the interference between the emission of FITC and CPs, was realized using singly-stained microspheres. Finally, APGMA-CP-BSA-FITC microspheres were found to be double positive for CP and FITC with very high percentage (>95%), suggesting the bioconjugation is very effective. This study provides a facile method for simultaneous introduction of fluorescence and reactive sites onto the micro spheres, which is very promising to be used as general strategy for fabricating fluorescence microspheres for application in high-throughput technology. (C) 2019 Elsevier Inc. All rights reserved.