The rampant hepatitis B virus (HBV) seriously endangers human health, and hepatitis B surface antigen (HBsAg) is its early diagnostic marker. Therefore, it is crucial to construct a fast and highly sensitive HBsAg detection method. Based on high-efficiency magnetic separation technology and fluorescent composite material labelling technology, an accurate, fast and sensitive fluorescent immunosensing system for HBsAg detectionwas developed. Immunomagnetic beads constructed from carboxyl-functionalized Fe3O4 nanoparticles (Fe3O4-COOH) with excellent magnetic response performance were used as efficient capture carriers for HBsAg. Immunofluorescence composite microspheres constructed based on ultra-stable polystyrene-coated CsPbBr3 perovskite nanocrystals (CPB@PSAA) with high hydrophilic properties, were excellent fluorescent markers for HBsAg. Using thissensitive sandwich fluorescence sensing system a good linear relationship within the range of 0.2-15ng/mL was established between HBsAg concentrationand fluorescence intensity with alimit of detection (LOD) of 0.05ng/mL. Thesystem obtained satisfactory results when tested on real human serum samples. The magnetic-assisted fluorescence immune-sandwich sensor system has broad application prospects in biomedicine such as rapid and early diagnosis and effective prevention of infectious diseases.