In the quantum dot-encoded microspheres based on the suspension array technology, the nonspecific biofouling will decrease the detection sensitivity and the multiplex detection ability. In order to inhibit the nonspecific biofouling, the polyethylene glycol grafted styrene maleic acid copolymer (PEG-g-PSMA) was used as substrate, and the CuInS₂/ZnS quantum dot-encoded PEG-g-PSMA fluorescent microspheres were fabricated via the Shirasu porous glass (SPG) membrane emulsification-emulsion solvent evaporation technique. The grafting ratio and the chain length of PEG in PEG-g-PSMA were controlled by adjusting the feed mass and relative molecular mass of methoxy polyethylene glycol (mPEG), respectively. The microspheres were further applied to the immunoassay of CA199. The results of morphology and fluorescence properties show that the prepared microspheres are spherical and monodisperse, with an average particle size of 5 μm, and the internal quantum dots and fluorescence distribution are uniform. Immunoassay shows that the microspheres can significantly inhibit the nonspecific adsorption. When the optimal grafting ratio of PEG is 30 and the relative molecular mass of PEG is 1000, the limit of detection (LOD) to CA199 reaches up to 0.9 kU/L (1 U=1 μmol/min). The PEG-g-PSMA fluorescent microspheres can be prepared in large quantites by this method and have a promising application in multiplex immunoassay.