With the application and development of a luminescent oxygen channeling assay (LOCI) in the field of bio-assay, the synthesis of phthalocyanine photosensitive polymer microspheres has received extensive attention. However, the non-specific adsorption of proteins by microspheres will affect the practical application of microspheres. In this study, an attempt was made to prepare PEG segment-functionalized polystyrene microspheres by one-step copolymerization of amphiphilic macromolecular monomers with polymerizable double bonds (PEOVB) and carboxyl monomers with styrene, followed by dissolving the photosensitizer into the microspheres. The microspheres were characterized using SEM, FT-IR spectroscopy and UV-vis spectroscopy. The results showed that the size of the microspheres was homogeneous. With the increase of PEOVB dosage, the non-specific adsorption capacity of microspheres decreased, and the adsorption capacity of BSA decreased from 28.1 mg g⁻¹ to 0.6 mg g⁻¹ (about 97.9% decline). The LOCI was used to detect ricin toxin (RT), and the donor microspheres prepared had the highest signal-to-noise (S/N) ratio (about 64.8), almost 7 times higher than that of PEOVB-free (about 9.7), when the PEOVB content was 10 wt%. The analytical performance of this system in detecting RT shows that the obtained donor microspheres (10 wt%PEOVB) can reach a detection limit of 0.1 ng mL⁻¹ and the linear concentration range of 0.1 to 50 ng mL⁻¹. The results indicate that the donor microspheres synthesized here are effective as microcarriers for LOCI.