question: [Image: see text] Direct detection of SARS-CoV-2 in biological specimens is often challenging due to the low abundance of viral components and lack of enough sensitivity. Herein, we developed a new type of chemiluminescent functionalized magnetic nanomaterial for sensitive detection of the SARS-CoV-2 antigen. First, HAuCl(4) was reduced by N-(aminobutyl)-N-(ethylisoluminol) (ABEI) in the presence of amino magnetic beads (MB-NH(2)) to generate ABEI-AuNPs, which were directly assembled on the surface of MB-NH(2). Then, Co(2+) was modified onto the surface to form MB@ABEI-Au/Co(2+) (MAA/Co(2+)). MAA/Co(2+) exhibited good chemiluminescence (CL) and magnetic properties. It was also found that it was easy for the antibody to be connected with MAA/Co(2+). Accordingly, MAA/Co(2+) was used as a sensing interface to construct a label-free immunoassay for rapid detection of the N protein in SARS-CoV-2. The immunoassay showed a linear range from 0.1 pg/mL to 10 ng/mL and a low detection limit of 69 fg/mL, which was superior to previously reported methods for N protein detection. It also demonstrated good selectivity by virtue of magnetic separation, which effectively removed a sample matrix after immunoreactions. It was successfully applied for the detection of the N protein in spiked human serum and saliva samples. Furthermore, the immunoassay was integrated with an automatic CL analyzer with magnetic separation to detect the N protein in patient serums and rehabilitation patient serums with satisfactory results. Thus, the CL immunoassay without a complicated labeling procedure is sensitive, selective, fast, simple, and cost-effective, which may be used to combat the COVID-19 pandemic. Finally, the CL quenching mechanism of the N protein in the immunoassay was also explored.