In this work, the chemiluminescence (CL) reagent luminol, the CL catalyst horseradish peroxidase (HRP) and antibody ternary codified gold nanoparticles (AuNPs) were assembled for an amplified CL immunoassay. Firstly, luminol and HRP bifunctionalized AuNPs (HRP–luminol–AuNPs) with good CL activity and catalytic properties were synthesized via a simple and facile strategy. Then, luminol, HRP and antibody ternary codified AuNPs (HRP/Ab–luminol–AuNPs) with good CL activity, catalytic properties, and antigen-specificity were further assembled. In the presence of the corresponding antigen, crosslinking aggregation of the HRP/Ab–luminol–AuNPs occurred due to the highly specific antigen–antibody immunoreactions, resulting in a great increase in CL intensity. Then, a label-free homogeneous CL immunoassay was developed by virtue of the HRP/Ab–luminol–AuNPs acting as an amplifying sensing probe. The fabricated immunosensor exhibited a wide linear range from 0.1 ng mL⁻¹ to 1 μg mL⁻¹ with a detection limit of 0.03 ng mL⁻¹ for human IgG determination. Furthermore, the sensing strategy was extended to the detection of the cancer biomarker alpha-fetoprotein (AFP) with a detection limit of 5 pg mL⁻¹, which is more sensitive than the conventional enzyme-linked immunosorbent assay (ELISA). The proposed sensing strategy is simple, sensitive, selective, low-cost and convenient, and could be used for the detection of antigen in real samples, so it holds great application potential in clinical diagnosis and biomedical applications. Moreover, this sensing strategy can serve as a general detection platform for the immunoassay of other biomolecules by using the respective antibodies and corresponding antigens.