In this study, mesoporous silica nanospheres (MSNs) are synthesized and biofunctionalized with streptavidin for the development of an ultrasensitive chemiluminescence (CL) immunoassay. The streptavidin-functionalized MSNs have a large specific surface area and exhibit excellent hydrophilicity, which are favourable to effectively capture biotinylated antibodies and retain their bioactivity. We used α-fetoprotein (AFP) as a model analyte, which can be detected in a wide linear range of 0.01–5.0 ng mL⁻¹ with a very low detection limit down to 9 pg mL⁻¹ (S/N = 3). The assay results of clinical human serum samples using the present method are in acceptable agreement with the reference values obtained from the hospital. This biofunctionalized MSN-based CL immunoassay provides a promising approach for early screening of tumor diseases.