In this paper, a simple and reproducible method to synthesize intense fluorescence and photostable silica nanoparticles has been developed. Firstly, silica nanoparticles were prepared based on the Stöber method, and hydrophobic europium chelates were adsorbed into their micro-holes by ultrasonication and phase-transfer solvent. Then they were modified by cationic polyelectrolyte polydiallyldimethylammonium chloride (PDAC) and anionic polyelectrolyte poly(acrylic acid) (PAA) layer by layer. Using them as detection probes and magnetic nanoparticles as capture probes, a novel immunoassay for determination of hepatitis B surface antigen (HBsAg) was proposed. Under the optimal experimental conditions, the immuno-probes performed with a good linear range over 10–200 ng mL⁻¹ with a calibration curve equation of y = −549.24 + 652.44x (R = 0.9901). Based on the hydrophobicity and intense fluorescence europium chelates absorbed into silica nanoparticles, one-step fluorescence immunoassay has been realized and may have potential applications in clinical assays.