Polymeric microparticles obtained via the emulsion confinement self-assembly of block copolymers have been used as the effective scaffold for construction of bioactive agents for various biomedicine applications, however the used block copolymers are normally synthesize via the controlled/living polymerization that involves tedious workload. Herein, we have discovered that a homo-polyarylene ether nitrile (abbreviated as homo-PEN), obtained via the industrially scalable step-growth nucleophilic substitution polycondensation, can be transformed into sub-micrometer sized polymeric microparticles via emulsion confined self-assembly. We found that the size of obtained microparticles was dependent on the molecular weight of homo-PEN or added ethanol content during self-assembly. More interestingly, the surface cyano of as-obtained polymeric microparticles can be partially converted into carboxyl groups after hydrolysis in alkaline aqueous solution without any morphology destruction. Consequently, the protein A was further covalently grafted on the carboxylated PEN microparticles to obtain the so-called AP beads that showed comparable antibody enrichment as that of commercial beads. Meanwhile, the co-assembly of homo-PEN with red-emitting fluorescent quantum dots (QD) leads to generation of the Q-beads exhibiting stable fluorescence both in aqueous solution and after internalized into macrophage cells, which can be explored for quantitative evaluation of macrophage phagocytosis activity via flow cytometry. In short, this work opens new way for fabrication of multifunctional bioactive microparticles using easily synthesized polyarylene ethers.